一株嗜酸乳杆菌突变株亚油酸异构酶的纯化及性质

亚油酸异构酶可以把亚油酸转化为共轭亚油酸。用硫酸铵沉淀、透析、凝胶过滤等步骤 ,从 1株嗜酸乳杆菌突变株中分离纯化了该酶。纯化倍数为 5 2 .0倍、比活力达 5 1 3 .0U/mg、活力回收 7.0 %。用SDS PAGE测得该酶亚基的分子量为 40 .7ku ;该酶的最适反应pH值为 4.0左右 ,最适反应温度为 3 0～ 40℃ ,在 pH 2 .0～ 7.0和 60℃以下较稳定。Fe2 + 、Mg2 + 、Zn2 + 、Na+ 能提高酶活性 ,Hg2 + 、Cu2 + 、Mn2 + 、Fe3+ 能抑制酶活性。以亚油酸为底物时该酶的动力学常数为 2 1 .6mmol/L。     

Efficient biosynthesis of D-allose from D-psicose by cross-linked recombinant L-rhamnose isomerase: separation of product by ethanol crystallization

Mass production of a rare aldohexose D-allose from D-psicose was achieved in a batch reaction by crude recombinant L-rhamnose isomerase (L-RhI) cross-linked with glutaraldehyde. The D-psicose substrate was, in turn, mass produced from a naturally abundant ketohexose D-fructose by immobilized recombinant D-tagatose 3-epimerase (D-TE). At an equilibrium state, 25% of D-psicose was isomerized to D-allose, that is, 25 g of D-allose was obtained from 100 g of D-psicose. The D-allose product was easily separated and crystallized from the reaction mixture that contains 25%D-allose, 8%D-altrose and 67%D-psicose using ethanol. Empirically, approximately 338 g, that is, 90% of a theoretical overall yield for the purification of pure D-allose crystals was produced from 1.5 kg of D-psicose within 30 d using a constructed bioreactor. The cross-linked enzyme had an operative half-life of two months after repeated usages.     

Streptomyces hygroscopicus谷氨酰胺转胺酶的分离纯化及其性质研究

将新筛选的吸水链霉菌生产的谷氨酰胺转胺酶发酵液抽滤、盐析、离心和冷冻干燥,制得粗酶产品,酶的比活力为5 4 0U/ g ,收率为77 90 %。对粗酶的研究发现,在2 5～4 0℃、pH 6 0～8 0范围内,该酶都有较高的反应活性和稳定性。粗酶经离子交换层析后得到了较高的纯化,纯化倍数为13 1倍,收率6 3% ;再经凝胶过滤层析,酶纯化了1 14倍,收率6 5 % ;经SDS -PAGE电泳检测,得到了单一的区带,根据电泳标准分子量计算,该酶的相对分子质量约为38 32 4ku。     

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.     

Production of high-fructose rice syrup and high-protein rice flour from broken rice

The objective of this study was to develop a process for the production of both high-fructose rice syrup and high-protein rice flour from broken rice. The rice flour was obtained from broken rice by using either a dry or wet milling method. The glucose produced from the slurry of various raw materials by treating with α-amylase and glucoamylase was compared. Results indicated that cassava and corn starch were better raw materials than rice flour. However, the filtered residue of liquefied rice slurry could be recovered as high-protein rice flour. The particle size of rice flour had a small effect on the glucose yield. The orthogonal-array table (L₂₇) method of experimental design was employed to determine optimum conditions for liquefaction. The glucose yield based on starch was 90.8±3.6% under the following optimum conditions α-amylase, 0.12%; rice flour, 20%; temperature, 96°C; time, 90 min. The filtrate from liquefied rice slurry was saccharified at 60°C with three different concentrations of glucoamylase. The higher the enzyme concentration, the shorter the time required to reach the maximum yield. After saccharification, the glucose solution was decolourised, desalted and concentrated to 40% d.s. and then isomerised to fructose at 60°C under continuous operation by using immobilised glucose isomerase packed in a column. The isomerised syrup was then purified and concentrated to 71% d.s. The final high-fructose rice syrup contained 50% glucose, 42% fructose and 3% maltose. After liquefaction, the rice slurry was centrifuged and the precipitate was dried by either spray or drum drying. The composition of these two high-protein rice flours was almost the same and the protein content was about three times as high as the raw material. There were significant differences in surface structure of rice flour and high-protein rice flours, as observed by the scanning electron microscope.     

Relationship among trans and conjugated fatty acids and bovine milk fat yield due to dietary concentrate and linseed oil

Effects on fatty acid profiles and milk fat yield due to dietary concentrate and supplemental 18:3n-3 were evaluated in 4 lactating Holstein cows fed a low- (35:65 concentrate:forage; L) or high- (65:35; H) concentrate diet without (LC, HC) added oil or with linseed oil (LCO, HCO) at 3% of DM. A 4 x 4 Latin square with four 4-wk periods was used. Milk yield and dry matter intake averaged 26.7 and 20.2 kg/d, respectively, across treatments. Plasma acetate and beta-hydroxybutyrate decreased, whereas glucose, nonesterified fatty acids, and leptin increased with high-concentrate diets. Milk fat percentage was lower in cows fed high-concentrate diets (2.31 vs. 3.38), resulting in decreases in yield of 11 (HC) and 42% (HCO). Reduced yields of 8:0-16:0 and cis9-18:1 fatty acids accounted for 69 and 17%, respectively, of the decrease in milk fat yield with HC vs. LC (-90 g/d), and for 26 and 33%, respectively, of the decrease with HCO vs. LCO (-400 g/d). Total trans-18:1 yield increased by 25 (HCO) and 59 (LCO) g/d with oil addition. Trans10-18:1 yield was 5-fold greater with high-concentrate diets. Trans11-18:1 increased by 13 (HCO) and 19 (LCO) g/d with oil addition. Trans13+14-18:1 yield increased by 9 (HCO) and 18 (LCO) g/d with linseed oil. Yield of total conjugated linoleic acids (CLA) in milk averaged 6 g/d with LC or HC compared with 14 g/d with LCO or HCO. Cis9,trans11-CLA yield was not affected by concentrate level but increased by 147% in response to oil. Feeding oil increased yields of trans11,cis13-, trans11,trans13-, and trans,trans-CLA, primarily with LCO. Trans10,cis12-CLA yield (average of 0.08 g/d) was not affected by treatments. Yield of trans11,cis15-18:2 was 1 g/d in cows fed LC or HC and 10 g/d with LCO or HCO. Yields of cis9,trans11-18:2, cis9,trans12-18:2, and cis9,trans13-18:2 were positively correlated (r = 0.74 to 0.94) with yields of trans11-18:1, trans12-18:1, and trans13+14-18:1, respectively. Plasma concentrations of biohydrogenation intermediates with concentrate or linseed oil level followed similar changes as those in milk fat. Milk fat depression was observed when HC induced an increase in trans10-18:1 yield. A correlation of 0.84 across 31 comparisons from 13 published studies, including the present one, was found among the increase in percentage of trans10-18:1 in milk fat and decreased milk fat yield. We observed, however, more drastic milk fat depression when HCO increased yields of total trans-18:1, trans11,cis15-18:2, trans isomers of 18:3, and reduced yields of 18:0 plus cis9-18:1.     

A novel enzymatic approach to the massproduction of L-galactose from L-sorbose

Wild-type strain of Pseudomonas cichorii ST-24 was unable to grow on D -psicose and inductively produced D -tagatose 3-epimerase (D -TE) with D -tagatose as an inducer. We have isolated a constitutive mutant, designated strain Ka75, which had acquired a new ability to grow on a mineral salts medium containing D -psicose as a sole carbon source. The D -psicose-metabolizing mutant synthesized a high level of D -TE. When grown on the culture medium supplemented with Mn(2+), the mutant strain produced around 250-fold higher activity than did the parent strain. Enzymatic properties of the constitutive enzyme were similar to those of the wild-type. Using the immobilized D -TE and recombinant L-rhamnose isomerase (L-RhI) from Escherichia coli strain JM109, a two-step enzymatic reaction was performed for massproduction of a rare aldo-hexose monosaccharide, L-galactose, from a common one, L-sorbose. In the first step, L-sorbose was epimerized to L-tagatose in a yield of 28%. The L-tagatose obtained was utilized as a starting material for L-galactose preparation by the immobilized L-RhI. At equilibrium, approximately 30% L-tagatose was isomerized to L-galactose. Finally, 7.5 g of L-galactose was obtained from 100 g of L-sorbose, viz an overall yield of 7.5%. The product obtained was purified and identified to be L-galactose by specific optical rotation and high performance liquid chromatography (HPLC) analysis, and was ultimately confirmed by (13)C nuclear magnetic resonance ((13)C NMR) and IR spectra.     

保加利亚乳杆菌诱导产亚油酸异构酶条件研究

亚油酸异构酶可由保加利亚乳杆菌经诱导产生,可以将亚油酸(LA)转化为共轭亚油酸(CLA)。本实验对诱导保加利亚乳杆菌产亚油酸异构酶的条件进行研究,利用紫外和气质联用仪(GC-MS)检测所生成的CLA。结果表明：在培养基中添加1.5‰(V/V) LA 时所产酶的共轭亚油酸转化率最高;温度为36℃,培养36h 为较适的培养条件;单独添加0.1%(m/V)的乳糖或0.1%(m/V)的氯化钠有利于诱导产酶;在培养基中直接添加LA 的效果优于培养3至12h 后再进行添加。诱导所产酶可将LA 转化为CLA,且含有9c,11t-CLA 异构体。     

Oil extractability from enzymatically treated goldenberry (Physalis peruviana L.) pomace: range of operational variables

Goldenberry pomace (seeds and skins) represents a large portion of the waste generated during juice processing (ca. 27.4% of fruit weight). The potential of goldenberry agro‐industrial wastes for use as raw material for production of edible oil was evaluated. Fruit pomace, contained 6.6% moisture, 17.8% protein, 3.10% ash, 28.7% crude fibre and 24.5% carbohydrates. The n‐hexane‐extractable oil content of the raw by‐products was estimated to be 19.3%. Aqueous enzymatic extraction was investigated for recovery of oil from the fruit pomace. The most significant factors affecting extraction were enzyme concentration, the time of digestion with enzymes, substrate concentration in water and the particle size of substrate. A broad variation in oil recovery was obtained depending on the operational conditions during the enzyme‐aided aqueous extraction. The optimum and economical values were those obtained for 4:0.02:1 water:enzyme:substrate ratio. Generally, enzymatic treatment increased the extraction yield. The more than 42% yield by enzymation compared with the nearly 3% yield in the control process (without enzyme) implies a significant increase in yield by about 92.8%. In single‐enzyme trials, cellulase EC gave the best yield. Although proteases slightly improve yield, the enhancement values are much lower than those obtained with Cellulase EC and Pektinace L40. Rapid increase in oil yield occurred as the enzyme concentration increased from 1 to 2 g/100 g substrate. Yield increased with dilution, but it began to fall when the substrate became more diluted. Moreover, extractability increased significantly when particle size reduced. Concerning the oil composition, there were no great changes in the fatty acid pattern of the oils extracted with different hydrolytic enzymes when compared with each other or to the solvent extracted oil. The main purpose of this study was to maximise the efficiency of the enzymatic treatment for oil recovery from goldenberry pomace. As a first step toward developing goldenberry as a commercial crop, the results provide important information for the industrial application of the fruit.     

无水乙醇萃取联合氧化铝柱层析制备高纯磷脂酰胆碱

以大豆粉末磷脂为原料,采用无水乙醇萃取和氧化铝柱色谱相结合的技术,研究了高纯度磷脂酰胆碱的制备方法。乙醇萃取后磷脂酰胆碱的纯度为62.00%,得率为31.94%。主要考察了柱层析过程中的固定相、洗脱液浓度、上样量、料液比及洗脱液流速对分离效果的影响。结果表明：当固定相为100～200 目三氧化二铝、洗脱液为90%乙醇、上样量为1 g/30 g、料液比为1∶12（g/mL）、洗脱液流速为3.0 mL/min时,磷脂酰胆碱的纯度可达到94.52%,回收率为83.71%。该研究结果为进一步探讨工业化制备磷脂酰胆碱的研究提供了技术支持和数据支撑。     

固定化亲和层析分离灵芝发酵液蛋白酶A抑制剂

建立了一种固定化亲和层析高效分离筛选蛋白酶A抑制剂的方法,在pH为4.0,戊二醛终浓度为0.6%,给酶量为40mg/0.2g壳聚糖的条件下,固定化酶的回收率较高,达45.9%;利用此固定化蛋白酶A(PrA)亲和柱一步分离灵芝发酵全粉抽提液中的PrA抑制剂,所得抑制剂与原两步层析法分离的PrA抑制剂GLPAI在分子量、糖与蛋白质比例及抑制特性上都较为相近。     

Dry matter intake and milk yield and composition of cows fed yeast prepartum and postpartum

Thirty-six multiparous and 12 primiparous Holstein cows were utilized in a completely randomized design to characterize the effects of feeding yeast cultures (Saccharomyces cerevisiae) and enzymes on dry matter intake and milk yield and composition. The prepartum diet consisted of a total mixed ration containing chopped grass hay, corn silage, and grain pellet. The postpartum diet consisted of a total mixed ration containing corn silage, legume silage, chopped legume hay, and grain pellet. Treatments consisted of 1) whey control, 10 g/d; 2) enzyme, 10 g/d; 3) yeast; 15 g/d; and 4) Biomate Yeast Plus (20 g/d; Chr. Hansen BioSystems, Inc., Milwaukee, WI). Treatments were top-dressed at feeding time. Cows were housed in a tie-stall barn, had continuous access to fresh water, and were fed once daily at 0800 h for ad libitum intake. Daily intake and orts were recorded beginning 28 d prior to the expected calving date through wk 13 of lactation. Daily milk yield and weekly milk samples were collected through wk 13 of lactation. Body weight and body condition score were recorded once every 2 wk throughout the experiment. Urine samples were collected at 30, 60, and 90 d of lactation and were analyzed for allantoin and creatinine. Least squares means for intake, milk yield, and milk composition were unaffected by treatment. The allantoin to creatinine ratio was not affected by treatment. Yeast cultures with or without enzyme had no direct effects on prepartum or postpartum dry matter intake or milk yield and composition.     

脱脂椰蓉可溶性膳食纤维制备工艺及单糖组成和理化特性分析

以脱脂椰蓉为原料,采用响应面分析法建立酶-化学法提取可溶性膳食纤维得率的二次多项数学模型,并验证数学模型的有效性。探讨酶添加量、酶解时间、碱添加量、碱解时间因素对可溶性膳食纤维得率的影响,优化提取工艺参数,确定最佳提取工艺参数为混合酶添加量0.5%、酶解时间50?min、碱液（NaOH溶液）质量分数5%、碱解时间40?min,在此条件下椰蓉粕可溶性膳食纤维得率达11.78%,持水性、持油性和膨胀性分别为3.8?g/g、5.2?g/g和3.1?mL/g。红外光谱分析发现,脱脂椰蓉可溶性膳食纤维处于缔合状态的氢键较多;高效液相色谱结果表明,可溶性膳食纤维含有9?种单糖,其中甘露糖、氨基半乳糖、半乳糖、阿拉伯糖含量较高,分别为537.21、40.38、39.48?mg/L和15.83?mg/L。     

一株嗜热菌木糖异构酶的纯化与性质研究

研究了1株嗜热菌(Anoxybacillus flavithermus)所产木糖异构酶的分离纯化以及酶学性质。结果表明,经硫酸铵沉淀、Sephadex G-75凝胶过滤、纤维素DE-52弱阴离子交换柱和Q Sepharose Fast Flow强阴离子交换层析得到的木糖异构酶,分子量约为181 ku,由4个相同分子量的亚基组成。酶反应的最适温度为80℃,最适为pH为7.0且最适pH范围宽泛,pH6.0~11.0酶反应活性能保持80%左右。该酶热稳定性及耐碱性能良好,70℃保温1 h后酶活仍能保持近80%左右;pH5.0~8.0保温1 h后酶活仍能保持近80%以上,甚至pH12.0保温1 h后酶活性仍能保持40%左右。Mn2+和Co2+对酶活性有明显促进作用,Zn2+、Cu2+以及Al3+对酶活性有一定程度的抑制。     

Purification and properties of an extracellular inulinase from Rhizopus sp. strain TN-96

A filamentous fungus, Rhizopus sp. strain TN-96, was isolated from rhizosphere soil samples. An extracellular inulinase was purified from the culture filtrate of strain TN-96 grown on inulin by DEAE-Cellulofine A-500 and Sephacryl S-200 HP chromatographies. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis, with an apparent M(r) of 83 kDa. The purified enzyme had specific activities of 17 U/mg toward inulin (I) and 0.32 U/mg toward sucrose (S) (I/S ratio, 53). Inulinase activity was optimal at pH 5.5 and 40 degrees C. The inulinase exhibited an apparent K(m) value of 9.0 mM for inulin. The enzyme also hydrolyzed raffinose, but not bacterial levan.     

假单胞菌H3壳聚糖酶的纯化及部分酶学性质

对 Pseudmona sp.H3产生的壳聚糖酶粗酶液采用(NH_4)_2SO_4盐析、Sephadex G-25脱盐、Sepharose Q-XL阴离子交换层析和Superdex G-75分子筛层析进行纯化,经SDS-PAGE鉴定为单蛋白带,分子质量约为33.8ku,酶反应最适温度为40℃,最适pH为5.0,降解壳聚精(D.A.90.14％)Km值为3.59g/L,V_(max)值为 3.80mmol/(L·min);Ba~(2+)、K~+、Co~(2+)对该酶有激活作用,而Zn~(2+)、Mn~(2+)、Al~(3+)、Cu~(2+)则对酶有抑制作用;此外,该酶除了能降解壳聚糖以外,还具有CMCase活性。     

酶解法提取罗非鱼血液中血红素的工艺条件优化

为更加科学有效地利用丰富的罗非鱼血液资源,研究酶解法提取罗非鱼血液中血红素的工艺条件,通过单因素试验和正交试验确定了复合酶提取血红素的最佳酶解条件为：温度40 ℃、pH 8.0、底物质量浓度6 g/100 mL、酶添加量8 000 U/g、酶解时间2 h。再经过热处理、酸沉淀和真空冷冻干燥等纯化工艺后得血红素终产品。在此条件下,血红素提取率达到80.9%,产品纯度为28.2%。该工艺简单可行,生产成本低,适合工业化生产,不仅能有效地减少环境污染,还能显著地增加企业产品的附加值。     

Abomasal infusion of oleic acid increases fatty acid digestibility and plasma insulin of lactating dairy cows

Our objective was to determine whether abomasal infusions of increasing doses of oleic acid (cis-9 C18:1; OA) improved fatty acid (FA) digestibility and milk production of lactating dairy cows. Eight rumen-cannulated multiparous Holstein cows (138 d in milk ± 52) were randomly assigned to treatment sequence in a replicated 4 × 4 Latin square design with 18-d periods consisting of 7 d of washout and 11 d of infusion. Production and digestibility data were collected during the last 4 d of each infusion period. Treatments were 0, 20, 40, or 60 g/d of OA. We dissolved OA in ethanol before infusions. The infusate solution was divided into 4 equal infusions per day, occurring every 6 h, delivering the daily cis-9 C18:1 for each treatment. Animals received the same diet throughout the study, which contained (percent diet dry matter) 28% neutral detergent fiber, 17% crude protein, 27% starch, and 3.3% FA (including 1.8% FA from a saturated FA supplement containing 32% C16:0 and 52% C18:0). Infusion of OA did not affect intake or digestibility of dry matter and neutral detergent fiber. Increasing OA from 0 to 60 g/d linearly increased the digestibility of total FA (8.40 percentage units), 16-carbon FA (8.30 percentage units), and 18-carbon FA (8.60 percentage units). Therefore, increasing OA linearly increased absorbed total FA (162 g/d), 16-carbon FA (26.0 g/d), and 18-carbon FA (127 g/d). Increasing OA linearly increased milk yield (4.30 kg/d), milk fat yield (0.10 kg/d), milk lactose yield (0.22 kg/d), 3.5% fat-corrected milk (3.90 kg/d), and energy-corrected milk (3.70 kg/d) and tended to increase milk protein yield. Increasing OA did not affect the yield of mixed milk FA but increased yield of preformed milk FA (65.0 g/d) and tended to increase the yield of de novo milk FA. Increasing OA quadratically increased plasma insulin concentration with an increase of 0.18 μg/L at 40 g/d OA, and linearly increased the content of cis-9 C18:1 in plasma triglycerides by 2.82 g/100 g. In conclusion, OA infusion increased FA digestibility and absorption, milk fat yield, and circulating insulin without negatively affecting dry matter intake. In our short-term infusion study, most of the digestion and production measurements responded linearly, indicating that 60 g/d OA was the best dose. Because a quadratic response was not observed, improvements in FA digestibility and production might continue with higher doses of OA, which deserves further attention.     

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%。     

Microbial production of inulo-oligosaccharides by an endoinulinase from Pseudomonas sp. expressed in Escherichia coli

In an attempt to utilize the whole cell as a biocatalyst for inulo-oligosaccharide (IOS) production from inulin, the endoinulinase gene (inu1) of Pseudomonas sp. was cloned into the plasmid pBR322 using EcoRI restriction endonuclease and Escherichia coli HB101 as the host strain. The endoinulinase from E. coli HB101/pKMG50 was constitutively expressed, producing a high yield of IOS (78%). In a batchwise reaction, the initial enzyme concentration determined the total oligosaccharide yield, and excess enzyme decreased the total oligosaccharide yield due to the formation of high amounts of free sugars such as glucose and fructose. The recombinant E. coli expressing endoinulinase activity were immobilized on a polystyrene carrier material, resulting in a dramatically enhanced thermal stability of the enzyme. Continuous production of IOS from inulin was also carried out at 50 degrees C using a bioreactor packed with the immobilized cells. Under the optimal operation conditions, continuous production of IOS was achieved with a productivity of 150 g/l.h for 17 d at 50 degrees C without significant loss of initial activity.