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Analyzing the change of DH, PCL and active calcium, and selecting each of best method. The results:(1) Dealing with the bone under high pressure ①High content of protein in sheep bone or bone soup:0.05MPa, 2h, 1:2;②Low amino acids in bone powder:0.13MPa, 4h, 1:3; Low amino acids in bone soup: 0.10MPa, 3h, 1:4;(2) Hydrolysis the bone soup with enzymes ①High content of calcium: neutral enzyme, 1500u/L, 6h;②High DH: pancreatic enzyme, 1:100, 6h;③ Low PCL: Pancreatic enzyme, 1:125, 6h; High PCL: alkalescency enzyme,

结果表明：A、高压①骨粉及骨汤蛋白质含量最高：压力为0.05MPa，高压2.0h，骨水比为1：2；②骨粉中氨态氮最低：压力0.13MPa，高压4.0h，骨水比为1：3；骨汤中氨态氮最低：0.10MPa，3.0h，骨水比1：4；B、酶解①游离钙含量最高：中性蛋白酶，酶浓度1500u/L，酶解6h；②水解度最高：胰蛋白酶，酶浓度1：100，酶解6h；③PCL值最低：胰蛋白酶，酶浓度1：125，酶解6h；PCL值最高：碱性蛋白酶，酶浓度1500u/L，酶解8h。

The results:(1) Dealing with the bone under high pressure ① High content of protein in sheep bone or bone soup:0.05MPa, 2h, 1:2;② Low amino acids in bone powder:0.13MPa, 4h, 1:3; Low amino acids in bone soup: 0.10MPa, 3h, 1:4;(2) Hydrolysis the bone soup with enzymes ① High content of calcium: neutral enzyme, 1500u/L, 6h;② High DH: pancreatic enzyme, 1:100, 6h;③ Low PCL: Pancreatic enzyme, 1:125, 6h; High PCL: alkalescency enzyme,

结果表明： A 、高压①骨粉及骨汤蛋白质含量最高：压力为0.05MPa ，高压2.0h ，骨水比为1：2；②骨粉中氨态氮最低：压力0.13MPa ，高压4.0h ，骨水比为1：3；骨汤中氨态氮最低：0.10MPa ，3.0h ，骨水比1：4； B 、酶解①游离钙含量最高：中性蛋白酶，酶浓度1500u/L ，酶解6h ；②水解度最高：胰蛋白酶，酶浓度1：100，酶解6h ；③ PCL 值最低：胰蛋白酶，酶浓度1：125，酶解6h ； PCL 值最高：碱性蛋白酶，酶浓度1500u/L ，酶解8h 。

The result of study showed:1 The effect of some inorganic urea inhibitor enzyme indicated boracic acid inhibits the activeness of urea enzyme in soil well, delayed the speed of urea hydrolysis in the soil and decreased the volatilization of ammonia , boracic acid,can be chosen as a inorganic urea enzyme inhibitor.2 The effect of some organic urea inhibitor enzyme showed that the dissociative Humic Acids of turfy soil and weathered coal inhibit the activeness of urea enzyme in soil well.

室内模拟试验，对几种缓释氮肥的释氮研究表明，和其它几种氮肥相比，PCF和PSF的控氮时间都较长。 6。盆栽试验表明，PCF的释氮效果比其它缓释氮肥的要好，且在油菜生育的各个时期，释氮都较充足，是适合油菜的良好缓释氮肥。

The main component of ramie glial was pectin, hemicellulose and lignin; The single factor on degumming of ramie test was respectively analyzed with a high vigorous of pectinase KDN and TZ-888 compound enzyme （mainly xylanase and mannanase on the pH value, bath ratio, dosage of enzyme, metal ions, temperature and time.The orthogonal test of L9（34 were done by bath ratio, dosage of enzyme, temperature and time. The results showed that the best compages of KDN pectinase on degumming was 1∶12 of bath ratio,pH 8.6,1 mmol/L Mg2+,300 IU/g of KDN pectinase,35℃、6 h;the best compages of TZ-888 compound enzyme on degumming was 1∶18 of bath ratio,pH 4.0,1 mmol/L Ca2+, 500 IU/g of TZ-888 compound enzyme,45 ℃,6 h.

摘 要：本研究根据苎麻胶质的主要成分为果胶、半纤维素和木质素，用高活性的KDN果胶酶和TZ-888复合酶（主要是木聚糖酶和甘露聚糖酶）从pH值、浴比、酶用量、金属离子、温度、脱胶时间等方面进行单因素试验，然后选取影响显著的浴比、酶用量、温度，时间进行四因素三水平的脱胶试验，结果表明鲜麻采用KDN果胶酶脱胶最佳组合为浴比1：18，pH 8.6， 1 mmoL/L Mg2+，酶用量300 IU/g，温度35 ℃、时间6 h时处理，TZ-888复合酶脱胶最佳组为浴比1：12，pH 4.0， 1 mmoL/L Ca2+，酶用量500 IU/g，温度45 ℃，时间6 h进行脱胶为优化的试验条件。

The main component of ramie glial was pectin, hemicellulose and lignin; The single factor on degumming of ramie test was respectively analyzed with a high vigorous of pectinase KDN and TZ-888 compound enzyme （mainly xylanase and mannanase on the pH value, bath ratio, dosage of enzyme, metal ions, temperature and time.The orthogonal test of L9（34 were done by bath ratio, dosage of enzyme, temperature and time. The results showed that the best compages of KDN pectinase on dry ramie degumming was 1∶12 of bath ratio,pH 8.6,1 mmol/L Mg2+,20 IU/g of KDN pectinase,4℃、4h;the best compages of TZ-888 compound enzyme on degumming was 1∶18 of bath ratio,pH 4.0,1 mmol/L Ca2+, 300 IU/g of TZ-888 compound enzyme,40 ℃,5 h.

摘 要：本研究根据苎麻胶质的主要成分为果胶、半纤维素和木质素，用高活性的KDN果胶酶和TZ-888复合酶（主要是木聚糖酶和甘露聚糖酶）从pH值、浴比、酶用量、金属离子、温度、脱胶时间等方面进行单因素试验，然后选取影响显著的浴比、酶用量、温度，时间进行四因素三水平的脱胶试验，结果表明干苎麻采用KDN果胶酶脱胶最佳组合为浴比1：12，pH 8.6， 1 mmoL/L Mg2+，酶用量200 IU/g，温度45 ℃、时间4 h时处理，TZ-888复合酶脱胶最佳组为浴比1：18，pH 4.0， 1 mmoL/L Ca2+，酶用量300 IU/g，温度40 ℃，时间5h进行脱胶为优化的试验条件。

Recombinant ptotein made up 39.2%(pET-20b-bglA) and 33.5%(pET-28a-bglA) of the total proteins in the intracellular fraction, the solubility proportion of the enzyme up to 32.8%(pET-20b-bglA) and 40.1%(pET-28a-bglA), the activities of the enzyme were 66.8 (pET-20b-bglA) and 71.2 U/mg (pET-28a-bglA). These results showed that E. coli BL21-CodonPlus (DE3)-RIL with argU tRNA, ileY tRNA and leuW tRNA genes helped to improve expression of the enzyme through enhanced identification of the rare codons AGA/AGG, AUA and CUA. Further optimized the conditions for inducing, the solubility proportion of the enzyme was 70.6% at 16 °C and 1.2 times higher than 37 °C. The solubility proportion of the enzyme increased from 12.3% to 32.8% when IPTG concentrations dropped from 1000 μM to 25 μM. The recombinant enzyme was purified by heat treatment, DEAE Sepharose anion-exchange chromatography and TOYOPEARL HW-55F.

maritima MSB8 的-葡萄糖苷酶基因 bglA克隆至表达载体 pET-20b 和 pET-28a，构建重组质粒 pET-20b-bglA 和 pET-28a-bglA，然后转化不同大肠杆菌 E.coli 宿主，Tm-SIGlA 在 E.coli BL21-CodonPlus(DE3)-RIL 中获得高效表达，重组蛋白的表达量为 33.5%(pET-28a-bglA)和 39.2%(pET-20b-bglA)，可溶性比例为 32.8%(pET-20b-bglA)和 40.1%(pET-28a-bglA)，比酶活达 66.8 (pET-20b-bglA)和 71.2 U/mg (pET-28a-bglA)，这些结果表明，E.coli BL21-CodonPlus(DE3)-RIL 宿主带有的 argU tRNA、ileY tRNA 和 leuW tRNA 基因，分别增强对稀有密码子 AGA/AGG、AUA 和 CUA 的识别，有助于提高该酶在 E.coli 中的表达；进一步优化诱导条件，重组 E.coli BL21-CodonPlus(DE3)-RIL/pET-20b-bglA 在 37 ℃下诱导培养，IPTG 浓度由 1000 μM 降至 25 μM，目的蛋白可溶性表达由 12.3%增至 32.8%，提高 1.7 倍，16 ℃下低温诱导实现目的蛋白 70.6%的可溶性表达，较 37 ℃下诱导培养提高 1.2 倍。

On the basis of the kinetic equation of substrate reaction in the presence of urea or guanidine hydrochloride, all microscopic kinetic constants for the free enzyme and enzyme-substrate binary and ternary complexes have been determined. The results of the present studies indicate that:①In the presence of urea or guanidine hydrochloride, enzyme-substrate complexes lose their activity less rapidly than the free enzyme. Therefore, both substrates, NADPH and 7, 8-dihydrofolate, protect dihydrofolate reductase against inactivation.②The denaturation of dihydrofolate reductase by urea follows single-phase kinetics, and changes in enzyme activity and tertiary structure proceed simultaneously in the unfolding process, so it may be an"all or none"process.③The GdnHCl-induced unfolding of the dihydrofolate shows a biphasic transition, while the change in the enzyme activity is a single exponential process. The rate constant of inactivation is consistent with that of the fast conformational change. Therefore, the kinetic intermediate of protein unfolding should be a partially folded and inactive form.

我们根据在脲或盐酸胍存在下的底物反应动力学方程求得游离酶和酶底物二元、三元复合物的微观动力学常数，结果表明：①酶-底物二元、三元复合物的失活速度明显慢于游离酶，说明两个底物二氢叶酸和NADPH对酶的失活都具有一定程度的保护作用；②在脲作用下，酶的失活和构象变化均为单指数项过程，而且酶的活力丧失和三级结构变化是同时发生的，说明二氢叶酸还原酶的脲变性可能是一个"全或无"的两态过程；③在盐酸胍作用下，酶的构象变化为两相过程，而失活则是单指数项过程，酶分子构象变化的快相速度常数与失活速度常数基本一致，因此我们认为二氢叶酸还原酶的盐酸胍变性过程中存在一个没有活力、但仍具备一定空间结构的变性中间体。

The experiment two: enzyme preparation significantly improved average daily gainand feed conversion ratio (P＜0.05). Enzyme preparation significantly increased energymetabolizability and digestibility of crude fiber, crude protein and neutral detergent fiber,but had no remarkable effect on digestibility of dry matter, crude fat and acid detergentfiber. Enzyme preparation significantly decreased the relative viscosity of duodenal andjejunal digesta. The pH of intestine had no noticed difference in all groups. Enzymepreparation significantly decreased relative weight of gizzard, proventficulus, duodenum,jejunum and ileum. Enzyme preparation significantly increased villus size of duodenumand jejunum, and villus to crypt ratio of duodenum and ileum significantly increased too.Enzyme preparation considerably decreased ileal crypt height (P＜0.05), and didn"t affectthickness of intestinal wall. Supplementing enzyme preparation, the serum glucose, totalprotein and alanine aminotransferase, but enzyme preparation hadn"t noticed influenceupon uric acid, total cholesterol, triglyceride and high-density lipoproteins. Enzymepreparation significantly increased insulin, triiodothyronine and insulin-like growthfactor-Ⅰ. Adding enzyme preparation, the percentage of thyroid stimulating hormone andgrowth hormone in the serum increased 16.44%, 19.18% and 18.84%, 21.74%respectively, and the percentage of glucagon and thyroxine decreased 12.07%, 14.36% and 13.79%, 15.40%, but failed to reach statistical significance (P＞0.05). Enzymepreparation significantly increased (P＜0.05) the trypsin and amylase activity of duodenaland jejunal digesta, but enzyme preparation didnt affect significantly (P＞0.05) theintestinal lipase activity and pancreatic digestive enzyme. Enzyme preparation had nosignificant effect on caecal microbial population.

试验二：酶制剂显著提高平均日增重和饲料转化率(P＜0.05)；酶制剂显著提高能量代谢率及粗纤维、粗蛋白、中性洗涤纤维消化率(P＜0.05)，而对干物质、粗脂肪、酸性洗涤纤维消化率影响不显著；酶制剂显著降低十二指肠和空肠食糜相对粘度(P＜0.05)；添加酶制剂对肠道pH影响不显著；酶制剂显著降低肌胃、腺胃、十二指肠、空肠、回肠相对重(P＜0.05)，显著提高十二指肠和空肠绒毛高度，显著增加十二指肠和回肠绒毛高度／隐窝深度，降低回肠隐窝深度(P＜0.05)，对肠壁厚度影响不显著；酶制剂显著提高血清葡萄糖、总蛋白和谷丙转氨酶浓度(P＜0.05)，对尿酸、总胆固醇、甘油三酯及高密度脂蛋白浓度影响不显著，显著提高胰岛素、T_3、IGF-Ⅰ水平，添加酶制剂后，促甲状腺激素、生长激素分别提高16.44％、19.18％和18.84％、21.74％，胰高血糖素和T_4分别降低12.07％、14.36％和13.79％、15.40％，但差异不显著；酶制剂对胰腺消化酶活性影响不显著，显著增加十二指肠和空肠胰蛋白酶、淀粉酶活性，对小肠脂肪酶活性影响不显著；酶制剂对盲肠微生物菌落数影响不显著。

Finally confirmed that,The thighbone and the osteoporosis morbidity is possibly connected protein 6, respectively are: The lactoferrin light chain, membrane association protein A3, the enolase, ATP gather the enzyme, the acetyl coenzyme A reductases, the myo calcium protein; The lumbar vertebra and the osteoporosis morbidity is possibly connected protein 5, respectively are: The actin, the keratin, the enolase, ATP gather the enzyme, the myosin; The thighbone and the strong bone valuable curative effect is possibly connected protein 9, respectively are: The enolase, ATP gather the enzyme, the myo-calcium protein, the creatine activating enzyme isozyme, the phosphoglyceric acid change flavor the enzyme, the myosin, the lactoferrin light chain, the pyruvic acid activating enzyme isozyme, the crown protein; The lumbar vertebra and the strong bone valuable curative effect are possibly connected protein 8, respectively are: The carbonic anhydrase, the actin, the αB-crystal protein, 3-phospho-glycerol aldehyde oxidase, the serum albumin, ATP gather the enzyme, the myosin, the enolase.

最后确认：股骨与骨质疏松发病可能相关的蛋白6个，分别为：乳铁蛋白轻链、膜联蛋白A3、烯醇化酶、ATP合酶、乙酰辅酶A还原酶、肌钙蛋白；腰椎与骨质疏松发病可能相关的蛋白5个，分别为：肌动蛋白、角蛋白、烯醇化酶、ATP合酶、肌球蛋白；股骨与强骨宝疗效可能相关的蛋白9个，分别为：烯醇化酶、ATP合酶、肌钙蛋白、肌酸激酶同工酶、磷酸甘油酸变味酶、肌球蛋白、乳铁蛋白轻链、丙酮酸激酶同工酶、冠蛋白；腰椎与强骨宝疗效可能相关的蛋白8个，分别为：碳酸酐酶、肌动蛋白、αB-晶体蛋白、3-磷酸甘油醛脱氢酶、血清白蛋白、ATP合酶、肌球蛋白、烯醇化酶。

Furthermore, the effects of different metallic salts,nitrogen sources,carbon sources,temperature,pH,water content and fermentation time on the enzyme activity and the yield of the acid protease were studied and the results were as follows: both MnCl2 and CuSO4 could activate the protease while other metallic salts inhibited the protease on different levels; addition of 1 % bean flour and 1 % glucose could increase enzyme yield and enzyme activity by 71 % and 31 % respectively; the maximal yield of enzyme was achieved under the conditions of temperature at 40 ℃ and pH as 7.0; water content and fermentation time would also influence enzyme yield on different levels.

不同金属盐对该酶酶活及不同氮源、碳源、温度、pH、含水量以及发酵时间对菌株产酶的影响试验结果表明：MnCl2，CuSO4均对该酶有激活作用，其他金属盐类对该酶有不同程度的抑制作用；添加黄豆粉（1 %）和葡萄糖（1 %）可使菌株产酶酶活分别提高71 %和31 %；培养温度为40 ℃以及培养基起始pH为7.0时产酶最高；含水量和发酵时间对产酶也有不同程度的影响。

According to the clear water experiment, aeration performance of the new equipment is good with high total oxygen transfer coefficient and oxygen utilization ratio.

曝气设备的动力效率在叶轮转速为120rpm～150rpm时取得最大值，此时氧利用率和充氧能力也具有较高值。

The environmental stability of that world - including its crushing pressures and icy darkness - means that some of its most famous inhabitants have survived for eons as evolutionary throwbacks, their bodies undergoing little change.

稳定的海底环境─包括能把人压扁的压力和冰冷的黑暗─意谓海底某些最知名的栖居生物已以演化返祖的样态活了万世，形体几无变化。