产菊糖酶克鲁维酶母Y-85的明胶固定化

克鲁维酵母Ｙ－８５的胞外菊糖酶占其总酶活的２８％,以１０％明胶包埋该酵母,酶活保留率为７３．９％。与游离细胞相比,固定化细胞菊糖酶的最适ＰＨ未改变,但当ＰＨ〈４和ＰＨ〉７时酶活稳定性更高;最适水解温度则升高了５℃,酶的热稳定性也有所提高。游离细胞的Ｋｍ值为９．３ｍｍｏｌ／Ｌ,固定化细胞则为１２．８ｍｍｏｌ／Ｌ。４℃贮存３０ｄ,固定化细胞酶活无损失,分批反应１０批次,固定化细胞酶活及机械强度保持良好     

一株中性蛋白酶产生茵的筛选及产酶条件优化

从酒曲中筛选出一株高产蛋白酶的菌株,命名为OPY6,初步鉴定为假单胞菌Pseudomonassp．,经Plackett．BurmanDesign设计和Box．Behnken响应面设计对其产酶培养基做了优化,最优产酶条件为：葡萄糖2．83％,淀粉0．87％,酵母膏0．55％,氯化钙0．001％,氯化钠0．5％,黄豆粉1％,初始pH=7．0,在最佳培养基添加量下蛋白酶活提高到134．718U／mL;又对该酶在水产蛋白降解过程中的酶解效果进行了评价。     

聚乙烯醇复合凝胶固定化糖化酶研究*

以聚乙烯醇复合凝胶作为载体固定化糖化酶,最终酶活达到1.558u/g干胶．酶活回收率是30．2％。该固定化酶在pH4.6,45℃下,以10%的可溶性淀粉为底物进行分批试验,操作半衰期为350h,具备高底物浓度下操作及贮存稳定性。     

海藻酸钠固定化β-葡萄糖苷酶的研究

以海藻酸钠为载体,研究了β-葡萄糖苷酶固定方法及其条件,并利用固定化β-葡萄糖苷酶进行了酶解试验。结果表明,采用交联-包埋方式,在海藻酸钠质量分数3．5％、给酶量100U／g载体、戊二醛体积分数1％、氯化钙质量分数2％的条件下固定β-葡萄糖苷酶2h,可以获得较佳的固定化效果。其固定率达到65％,重复分批利用20次仍能保持90％以上的酶解得率。利用固定化β-葡萄糖苷酶连续酶解纤维二糖时,在不同进料速度下有着不同的催化效率,当进料速度为1．5mL／min、1．0mL／min时,酶解得率分别达到96,7％和99．0％;与木霉纤维素酶协同水解纤维素时,在β-葡萄糖苷酶总酶活与滤纸酶活之比为0．5（FPA为2．0U／mL）的条件下,酶解滤纸纤维素和微晶纤维素60h的得率比单独采用木霉纤维素酶分别增加了20．4％和29．3％。研究结果对于解决酶法水解纤维资源得率低、酶使用成本高这一关键问题提供了一种有效的方法。     

植物乳杆菌多孔淀粉直接包埋工艺的研究

利用多孔淀粉的吸附特性,将植物乳杆菌包埋于多孔淀粉内,通过测定多孔淀粉对植物乳杆菌的包埋率,研究菌体浓度、多孔淀粉添加量、振荡转速、包埋温度、pH值、时间对包埋率的影响,确定最适包埋条件。结果表明:菌体浓度10~8cfu/mL,多孔淀粉添加量为2%,pH 6.0、20℃,200 r/min振荡处理40 min,在此条件下包埋率为79.5%,对包埋后的菌体进行喷雾干燥试验,其存活率较未包埋的从0.35%提高到29.5%。     

植酸酶YiAPPA与生淀粉结合域SBD融合酶的构建及酶学性质分析

旨在获得酶学性质改良的植酸酶YiAPPA与生淀粉结合域SBD的融合酶。通过在植酸酶YiAPPA的C末端融合嗜热酸性α-淀粉酶GTamy的生淀粉结合域SBD,获得融合酶YiAPPA-SBD。酶学性质分析表明,YiAPPA-SBD的高温活性和热稳定性得到了提高,并获得了对生玉米淀粉的结合能力。其中YiAPPA-SBD于55-90℃范围内的相对酶活均高于YiAPPA的相对酶活;于80℃的半衰期提高约2倍;在生玉米淀粉浓度大于8%的条件下,YiAPPA-SBD对其结合率达到80%以上。并且YiAPPA-SBD保留有YiAPPA的其它优良酶学性质,最适反应pH为4.5,37℃的绝对酶活高达3 900 U/mg,具有优良的pH稳定性和蛋白酶抗性。     

植酸酶YiAPPA与生淀粉结合域SBD融合酶的构建及酶学性质分析北大核心CSCD

旨在获得酶学性质改良的植酸酶YiAPPA与生淀粉结合域SBD的融合酶。通过在植酸酶YiAPPA的C末端融合嗜热酸性α-淀粉酶GTamy的生淀粉结合域SBD,获得融合酶YiAPPA-SBD。酶学性质分析表明,YiAPPA-SBD的高温活性和热稳定性得到了提高,并获得了对生玉米淀粉的结合能力。其中YiAPPA-SBD于55-90℃范围内的相对酶活均高于YiAPPA的相对酶活;于80℃的半衰期提高约2倍;在生玉米淀粉浓度大于8%的条件下,YiAPPA-SBD对其结合率达到80%以上。并且YiAPPA-SBD保留有YiAPPA的其它优良酶学性质,最适反应pH为4.5,37℃的绝对酶活高达3 900 U/mg,具有优良的pH稳定性和蛋白酶抗性。     

海藻酸钠包埋法制备固定化菠萝蛋白酶

以海藻酸钠为载体,包埋法固定菠萝蛋白酶,对固定化奈件进行优化,同时探讨固定化菠萝蛋白酶的部分酶学性能。结果表明：固定化菠萝蛋白酶的质量受海藻酸钠质量分数、固定化酶量、固定化时间以及CaCl2质量分数的影响,其最佳固定化条件为：海藻酸钠质量分数1．0％,CaCl2质量分数3％,固定化酶液量与海藻酸钠体积之比1：2,固定化时间60min,在此条件下,制备的固定化菠萝蛋白酶的比活力为211．8U／g（湿质量载体）,由此制得的固定化酶的最适pH为7．6,与游离酶相比,升高了0．8个pH单位,同时显示固定化菠萝蛋白酶能耐受较高的碱性环境,固定化酶最适温度与游离酶相同,均为50℃,固定化酶在较高温度范围内,仍能保持较高的相对活力。     

固定化细胞拆分DL-泛解酸内酯的初步研究

用卡拉胶包埋串珠镰孢霉菌Fusarium moniliforme SW-902菌丝体,得到D－泛解酸内酯水解酶活力较高的固定化细胞。与游离细胞相比较,固定化细胞酶活随pH变化的范围以及对温度适应的范围大致与游离细胞相仿。用固定化细胞进行反复分批酶水解30批,每天一批,酶活稳定,平均水解率28．0％。固定化细胞冰箱（4℃）贮存8周,酶活未见下降。     

真空微波诱变结合化学诱变选育酸性纤维素酶高产菌株

以酸性纤维素酶产生菌绿色木霉（Trichoderma viride）WL0512作为原始出发菌株,首先经自然分离筛选出一株产酶较稳定的菌株TVN-18,其羧甲基纤维素酶活（CMC酶活）达2765．8U／g,滤纸酶活（FPA酶活）达48．5U／g。再经真空微波和甲基磺酸乙酯（EMS）逐级诱变处理,获得了一株高产、稳产酸性纤维素酶的E6—1菌株,其CMC酶活达4396．6U／g,FPA酶活达126．0U／g,分别是菌株TVN-18的1．59倍和2．60倍。通过对固态发酵培养基麸皮和稻草比例、料水比以及初始pH值的优化,突变株的产酶能力进一步得到提高,其产的CIVIC酶活和FPA酶活分别提高了22．3％和22．4％。     

Phytohormone-mediated transformation of sugars to starch in relation to the activities of amylases, sucrose-metabolising enzymes in sorghum grain

Indole-acetic acid (IAA) and abscisic acid (ABA) were fed throughcomplete liquid medium (containing 2, 4, 8% sucrose) to detached earheads of sorghum. The effect of these phytohormones on interconvertion ofsugarsand their transformation to starch in relation to the activities of-, -amylases, sucrose-synthase (synthesis), sucrose-phosphatesynthase and soluble invertases was studied in the grain. This effect on theuptake of (U-¹⁴C) sucrose by detached ear heads and incorporation of¹⁴C into free sugars and starch of grain and into free sugars ofinflorescence parts was also studied. At concentrations of up to 4%sucrose in the culture medium, IAA increased the content of total free sugarsinthe grain. However, accumulation of starch and activities of - and-amylases increased when lAA was present even beyond the 4%sucroseconcentration in the culture medium. At all sucrose concentrations, the effectsof ABA and IAA were opposite. With 4% sucrose, both phytohormones causedmaximum accumulation of starch in the grain. ABA enhanced the relativeproportion of sucrose in the sugar pool with a concomitant reduction in theactivities of soluble acid (pH 4.8) and neutral (pH 7.5) invertases. Incontrast, IAA decreased the sucrose proportion of grain sugars with asimultaneous elevation and reduction in the activities of invertases andsucrose-phosphate synthase, respectively. Irrespective of sucrose concentrationin the culture medium, the activity of sucrose synthase (synthesis) wasenhancedwith IAA as well as ABA at their 10 M concentration. IAA alsoenhanced incorporation of ¹⁴C from (U-¹⁴C) sucrose intothe EtOH extract (principally constituted by free sugars) and starch of thegrain, but ABA caused the reverse effect. Based on the results, it is suggestedthat IAA and ABA have contrasting effects on the transformation of sucrose tostarch in sorghum grain where its capacity to synthesise starch is modulatedpositively by IAA and negatively by ABA.     

Partitioning of shoot and root dry matter and carbohydrates in bean plants suffering from phosphorus, potassium and magnesium deficiency

The influence of varied supply of phosphorus (10 and 250 mmolP m^–3) potassium (50 and 2010 mmol K m^–3) and magnesium(20 and 1000 mmol Mg m^–3) on the partitioning of dry matterand carbohydrates (reducing sugars, sucrose and starch) betweenshoots and roots was studied in bean (Phaseolus vulgaris) plantsgrown in nutrient solution over a 12 d period. Shoot and rootgrowth were quite differently affected by low supply of P, K,and Mg. The shoot/root dry weight ratios were 4.9 in the control(sufficient plants), 1.8 in P-deficient, 6.9 in K-deficientand 10.2 in Mg-deficient plants. In primary (source) leaves,but not in trifoliate leaves, concentrations of reducing sugars,sucrose and starch were also differently affected by low nutrientsupply. In primary leaves under K deficiency and, particularlyMg deficiency, the concentrations of sucrose and reducing sugarswere much higher than in control and P-deficient plants. Magnesiumdeficiency also distinctly increased the starch concentrationin the primary leaves. In contrast, in roots, the lowest concenfrationsof sucrose, reducing sugars and starch were found in Mg-deficientplants, whereas the concentrations of sucrose and starch wereparticularly high in P-deficient plants. There was a close relationshipbetween shoot/root dry weight ratios and relative distributionof total carbohydrates (sugars and starch) in shoot and roots.Of the total amounts of carbohyd rates per plant, the followingproportions were parti tioned to the roots: 22.7% in P-deficient,15.7% in control, 3.4% in K-deficient and 0.8% in Mg-deficientplants. The results indicate a distinct role of Mg and K in the exportof photosynthates from leaves to roots and suggest that alterationin photosynthate partitioning plays a major role in the differencesin dry matter distribution between shoots and roots of plantssuffering from mineral nutrient deficiency. Key words: Bean, carbohydrates, magnesium nutrition, phosphorus nutrition, potassium nutrition, shoot/root growth     

C]Sucrose Uptake and Labeling of Starch in Developing Grains of Normal and segl Barley

Previous work showed that the segl mutant of barley (Hordeum vulgare cv Betzes) did not differ from normal Betzes in plant growth, photosynthesis, or fertility, but it produced only shrunken seeds regardless of pollen source. To determine whether defects in sucrose uptake or starch synthesis resulted in the shrunken condition, developing grains of Betzes and segl were cultured in [¹⁴C]sucrose solutions after slicing transversely to expose the endosperm cavity and free space. In both young grains (before genotypes differed in dry weight) and older grains (17 days after anthesis, when segl grains were smaller than Betzes), sucrose uptake and starch synthesis were similar in both genotypes on a dry weight basis. To determine if sucrose was hydrolyzed during uptake, spikes of Betzes and segl were allowed to take up [fructose-U-¹⁴C]sucrose 14 days after anthesis and the radioactivity of endosperm sugars was examined during 3 hours of incubation. Whereas less total radioactivity entered the endosperm and the endosperm cavity (free space) of segl, in both genotypes over 96% of the label of endosperm sugars was in sucrose, and there was no apparent initial or progressive randomization of label among hexose moieties of sucrose as compared to the free space sampled after 1 hour of incubation. We conclude that segl endosperms are capable of normal sucrose uptake and starch synthesis and that hydrolysis of sucrose is not required for uptake in either genotype. Evidence suggests abnormal development of grain tissue of maternal origin during growth of segl grains.     

Carbon allocation in developing spruce needles. Enzymes and intermediates of sucrose metabolism

In lyophilized needles of Norway spruce ( Picea abies [L.] Karsten) and starting from bud break, we determined enzyme activities (sucrose phosphate synthase [SPS; EC 2.4,1.14]. sucrose synthase [SS; EC 2.4,1.13]. acid invertase [AI; EC 3.2,1.26]) and intermediates (starch, sucrose, glucose, fructose; fructose 6-phosphate, fructose 2.6-bisphosphate [F26BP]) of carbohydrate metabolism together with needle weight, shoot length, chlorophyll and protein. For up to 110 days after bud break, samples were taken twice a week from about 25-year-old trees under field conditions. At least three periods can be distinguished during needle maturation. During the first period (up to 45 days after bud break) Al showed the highest extractable activity. This coincided with very high levels of F26BP (up to 11 pmol [mg dry weight]⁻¹) and a transient increase of starch in parallel to a decrease of sucrose. The interval between 45 and 70 days after bud break was characterized by high SS activity (ratio of fructose/glucose >1), much decreased levels of F26BP (down to below 1 pmol [mg dry weight]⁻¹), and a pronounced increase in the dry weight/fresh weight ratio. In parallel, starch declined and soluble carbohydrates increased. Finally, needle maturation was characterized by decreasing SS and continuously increasing SPS activities, so that the ratio of SPS/SS increased more than 6-fold. AI. however, did not decline with maturation. Changes in pool sizes of metabolites and enzyme activities (AI. SPS) are consistent with current concepts on sink/source transition. SS is obviously important with regard to the synthesis of structural polysaccharides.     

Enhanced phytase production from Achromobacter sp. PB‐01 using wheat bran as substrate: Prospective application for animal feed

This article deals with the optimization of the various parameters for production of phytase using Achromobacter sp. PB‐01 in submerged fermentation (SmF). A semisynthetic medium containing ingredients of phytase screening media (PSM) supplemented with 2% (w/v) sucrose, 1% (w/v) peptone, and 10% (w/v) wheat bran was found to be the best production medium among the various combinations tried. Among various surfactants added to SmF, Triton X‐100 (0.1%) exhibited a 16% increase in phytase activity. An overall 11.2 fold enhancement in enzyme activity (0.79 U/mL→8.84 U/mL) was attained when SmF was carried out using 0.5% (v/v) inoculum of a 15 h old culture of Achromobacter sp. PB‐01 at an initial pH of 5.5, temperature 30°C and allowed to grow for 48 h. Presence of accessory hydrolytic enzymes in the crude extract further added value as feed additive by mediating efficient degradation of non‐starch polysaccharides (NSP). In addition, we also investigated the efficacy of phytase on different agro‐industrial residues using in vitro experiments that simulated the conditions of the digestive tract. Results indicate that phytase from our source hydrolyze phytate efficiently with the concomitant liberation of inorganic phosphate, protein, reducing sugar, and calcium. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Sesame hairy root cultures for extra-cellular production of a recombinant fungal phytase

Sesame (Sesamum indicum L.) hairy roots were transformed with a fungal (Aspergillus) phytase and their culture conditions were surveyed for the extra-cellular production of the recombinant phytase protein in shake flasks. Kanamycin resistance of sesame hairy roots was observed at 50 μg ml⁻¹ kanamycin sulfate and southern hybridization analysis confirmed the existence of the phytase gene in the hairy root genomic DNA. The continuous dark condition was more effective for both the root growth and phytase production than light. Slightly higher root growth was determined at 30 °C than 26 °C in Murashige & Skoog (MS) medium supplemented with 3% sucrose, while the final phytase production was greatest in MS medium with 5 or 3% sucrose at both temperatures of 26 and at 30 °C. Among the culture media used, full-strength MS medium was exclusively efficient for production of the recombinant phytase. Most rapid increase rates in both the root growth and phytase production were detected at the 4th week of the culture periods and thereafter their rates began to decrease. Our results indicated that 5–6-week culture periods may be necessary for the maximal phytase production. Western analysis revealed that even though the phytase proteins expressed were measured with greater activities in the liquid medium than in the root tissues, they were still retained in the tissues.     

Dry weight accumulation and starch-synthesis enzymes in grain of cultured ears of three winter wheat varieties

Detached ears of three winter wheat ( Triticum aestivum L.) varieties were cultured in solution for 12 days with sucrose levels varying from 36.5 to 292 m M. The dry weight and starch content of grains increased asymptotically with the sucrose level in the solution. At 4 days of culture, glucose phosphate isomerase (EC 5.3.1.9) activity grain⁻¹ was lower with 36.5 m M than with higher sucrose levels in the medium; at 8 days, adenosinc diphosphoglucose pyrophosphorylase (EC 2.7.7.27) and (soluble plus bound) starch synthase (EC 2.4.1.21) activities grain⁻¹ were higher with 146 and 292 m M sucrose than with 36.5 and 73 m M sucrose. The multiple regression of starch content over these enzyme activities showed that starch synthase was relatively more important as an independent variable. The dry weight and starch content of grains were higher in the variety Maris Huntsman than in Splendeur and Hobbit. The water content of grains was lower in Splendeur than in the other two varieties. At 4 days the glucose phosphate isomerase, adenosine diphosphoglucose pyrophosphorylase and starch synthase activities grain⁻¹ were smaller in Splendeur than in Hobbit and Maris Huntsman and al 8 days they were higher in Maris Huntsman than in Hobbit and Splendeur. The varietal differences in starch content of grains were related to the activities of glucose phosphate isomerase and especially of starch synthase.     

Effects of Culture Age, Washing and Storage Conditions on Desiccation Tolerance of Colletotrichum truncatum Conidia

Colletotrichum truncatum conidia produced from a one week-old culture in a liquid semi-defined medium with a C:N ratio of 5:1 were more tolerant of desiccation than those harvested from two or three week-old cultures. Conidia washed with 20% (w/v) sucrose germinated better than unwashed conidia or those washed in 10% (w/v) sucrose, 10 and 20% (w/v) glucose or fructose, 0.1% (w/v) soluble starch, 0.9% (w/v) NaCl or deionized water. Washing with sucrose (20% w/v) also resulted in significantly longer germ tubes than those produced by unwashed conidia or conidia washed with deionized water or NaCl (0.9% w/v). Conidia washed twice in sucrose showed greater desiccation tolerance during storage at 15% relative humidity (RH) and 15°C than at 30% RH and 15 or 25°C or at 15% RH and 25, 5 or -10°C.     

Evaluation of microencapsulation of a Bifidobacterium strain with starch as an approach to prolonging viability during storage

AIMS: To optimize a spray coating process for the production of encapsulated microspheres containing viable Bifidobacterium cells and to determine whether the readily gelatinized modified starch coating used in this study improved bacterial survival in foods or under acid conditions. METHODS AND RESULTS: An air inlet temperature of 100 degrees C was demonstrated to be optimal for the spray drying process, as it afforded good drying, low outlet temperatures (45 degrees C) and resulted in less than 1 log reduction in bifidobacteria numbers during drying. Maximum recovery yields of 30% were obtained after optimizing the air aspiration conditions. The average size of the Bifidobacterium PL1-containing starch microparticles was determined by scanning electron microscopy to be of the order of 5 microm. The starch-coated cells did not display any enhanced viability compared with free PL1 cells when exposed to acid conditions for 6 h or in two dry food preparations over 20 d storage at ambient temperature (19-24 degrees C). Determination of 1491 nucleotides of the 16S rRNA gene from PL1 indicated that it shared 97% homology with a previously sequenced Bifidobacterium ruminantium strain. CONCLUSIONS: Our data demonstrated that, although spray drying is a valuable process for encapsulating bifidobacteria, further work is required to ascertain a more appropriate coating material that will protect this strain against adverse environmental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of small, uniformly coated microspheres containing viable bifidobacteria using an affordable and industrially convenient process, such as spray drying, has commercial implications for the production of probiotic products. Although popular for use as a coating polymer by the food industry, this study indicated that modified starches might not be suitable for use as an encapsulating material for probiotic strains.