胰脏综合利用研究

用中性乙醇从猪胰脏提取了胰岛素和较高活力的胰酶。并从胰岛素盐析母液中制取了抑肽酶。粗品胰岛素的收率为2176±521U／kg胰脏；胰酶收率为8．44％±1．72％；抑肽酶收率为127±15kU／kg胰脏。     

珠母贝氨基多糖的分离纯化及其抗肿瘤活性的初步研究

探讨珠母贝氨基多糖的分离纯化及其抗肿瘤活性。马氏珠母贝全脏器经胰酶和枯草杆菌蛋白酶双酶水解、醇沉后得到氨基多糖粗品(CPG)．再经DEAE-52-纤维素柱纯化，并采用MTT法检测其体外抗肿瘤活性。结果表明：CPG和经DEAE-52-纤维素柱纯化级分GAG-1，GAG-2中总氨基己糖的质量分数分别为45．9％，56．5％和59.2％，级分GAG-1硫酸基的质量分数高达13.8％；体外抗肿瘤试验表明CPG，GAG-1和GAG-2具有抑制肿瘤细胞生长的作用，对HL-60细胞的抑制率分别为54．6％。50.0％和35．4％，且能显著增敏化疗药物5-Fu抑制肿瘤细胞的作用，其中GAG-1为主要的活性成分。     

乌贼墨糖胺聚糖的制备与理化性质研究

本实验采用胰蛋白酶、链霉蛋白酶两次酶解，乙醇沉淀和DEAE一纤维素离子交换柱分离，从新鲜无针乌(Sepiella maindroni de Rochebrune)墨中提取得到乌贼墨糖胺聚糖(GAG)；经醋酸纤维素薄膜电泳和聚丙烯酰胺凝胶电泳证明乌贼墨GAG为均一性多糖；紫外扫描无蛋白质的特征吸收峰；红外光谱分析，样品具有典型的GAG吸收峰；对该GAG酸水解产物的薄层色谱、纸色谱研究表明该GAG主要由氨基半乳糖、葡萄糖醛酸、岩藻糖3种单糖组成，含量分别为25．6％，29．2％，23．3％；硫酸根和蛋白质含量分别为8．2％，3．5％。     

不同酶源水解猪血液的研究

用细菌蛋白酶166、3942与胰酶对猪血进行水解比较,测定氨基氮、总氮含量,结果表明:细菌蛋白酶3942的氨基氮5.55％（以干物质计）,混合酶为6.28％,总氮为12.99％。用细菌蛋白酶3942或细菌蛋白酶166与3942的混合酶可以代替胰酶。     

胰性脑病的临床研究(附13例报告)

本组114例重症急性胰腺炎（SAP）患者，13例并发胰性脑病（PE），发生率为11．4％。其中早期PE（SAP发生后7日之内）8例，迟发PE（SAP发生7日以后）5例。3例行保守治疗，10例接受手术处理。1例存活，其余12例死亡。临床及实验研究表明，多种胰酶尤其是磷脂酶A2（PLA）在PE发病机理中起重要作用。     

蜈蚣粉酶解工艺最佳用酶的初步筛选

目的分别利用胃蛋白酶、胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶对蜈蚣粉进行酶解并选取最佳用酶来制备抗肿瘤多肽。方法利用4种酶对蜈蚣粉进行酶解,采用MTT体外抗肿瘤实验方法,以蛋白质的水解度及酶解提取液对乳腺癌MCF-7细胞的抑制率为指标,同时以蜈蚣粉的水溶性蛋白质含量为标准,评价4种酶对蜈蚣粉的酶解效果并筛选出最佳用酶。结果胃蛋白酶对蜈蚣粉的水解度为10.44%;胰蛋白酶对蜈蚣粉的水解度为28.29%,在0.125 mg·m L~(-1)及0.25 mg·m L~(-1)的较小浓度下对乳腺癌MCF-7细胞的抑制率低于对照组,但随浓度的增大抑制率逐渐增大,在0.5~2 mg·m L~(-1)浓度时,胰蛋白酶在4种酶中达到最大抑瘤效果;胰凝乳蛋白酶和弹性蛋白酶的水解度分别为50.45%、44.16%,当浓度>0.5 mg·m L~(-1)时其对乳腺癌MCF-7细胞的抑制率高于对照组,在浓度为0.5mg·m L~(-1)时抑制率分别为2.86%、36.37%,且随浓度增大抑制率增大。结论蜈蚣粉酶解工艺最佳用酶为胰蛋白酶,其次是胰凝乳蛋白酶和弹性蛋白酶,而胃蛋白酶活性较低可以去除。     

溶媒耐受性绿脓假单胞菌PseA产生耐溶媒蛋白酶的培养条件优化及产物鉴定

据报道，从源自Pseudomonas aeruginosa的溶媒耐受性绿脓假单胞菌株（PseA）中可获得耐溶媒蛋白酶。本研究所获得产生该酶的优化培养条件：以0．7％甘油为碳源，0．4％酪蛋白和0．6％酵母抽提物作为氮源，pH7．0；种子液培养36h后以2．5％的接种量接种；30℃振荡（250r／min）培养24h。在该条件下，菌体生长最好，酶活力最高（160lu／ml）。该蛋白酶的最适pH与温度为9．0和55℃。     

豆粕的不同酶解液发酵苏云金芽孢杆菌对Zwittermicin A产量的影响

目的 研究培养基中豆粕所含蛋白质的不同存在形式对zwittermicin A产量的影响.方法 豆粕经过3种不同的复合蛋白酶(碱/中蛋白酶、中性蛋白酶、酸/中蛋白酶)处理后形成了蛋白质、寡肽和氨基酸含量均不同的3种酶解液,即DPI,DP2和DP3.以苏云金芽孢杆菌库斯塔克亚种(Bacillus thuringiensis sp.kurstaki D1-23)为菌种,分别将这3种豆粕酶解液作为其氮源进行发酵.结果 从含有DP1的培养基D-1 中获得的苏云金芽孢杆菌生物量最低;含有DP2的培养基D-2中的zwittermicin A含量最高;从含有DP3的培养基D-3中得到的生物量最大.结论 氮源的存在形式不同,对Bacillusthuringiensis sp.turstaki D1-23 菌体繁殖和其次级代谢产物zwittermicin A的合成有不同影响.     

胆胰肠结合部医源性损伤的外科诊治分析

目的：研究胆胰肠结合部损伤的外科诊治方法及效果。方法回顾性分析29例胆胰肠结合部损伤患者的诊治情况。采用简单而短时间的手术充分清理和引流损伤局部及感染灶,彻底分流胆汁、胃肠液和胰液,控制胰酶的激活。结果29例胆胰肠结合部损伤患者均放置双套管引流,术后均给予营养支持及抑制胰腺分泌治疗。手术时间为（224．8±52．8）min,出血量为（82．6±59．4）mL,排气时间为（3．4±1．2）d,住院时间为（15．3±8．9） d,感染死亡1例（2．9％）。结论胆胰肠结合部损伤是手术中发生的医源性损伤,术中易漏诊,应根据术中损伤的单纯性或复合性及术后发现的具体情况进行相应的治疗。     

甘蔗渣纤维素固定化木瓜蛋白酶及其应用研究

匀浆状甘蔗渣纤维素用NaOH活化后，顺次经过高碘酸钠、尿素、甲醛处理，成为高反应性固定化酶载体，可用于木瓜黄蛋白酶的固定化，其酶蛋白的结合量与甲醛处理时间、酶固定时间、溶液酶浓度等有关，甲醛处理最适时间为14h，酶固定8h后，载体结合酶量趋向稳定；溶液酶最佳浓度范围为1－2．0mg/ml粗酶，固定化酶活力回收达34．5％，半衰期35天，固定化酶和溶液酶的Km值（底物酪蛋白，w/v,%)分别为0．12％和0．26％；固定化酶和溶液酶的最适PH分别为PH8．0和PH8．5，二者的最适温度均为60－70℃。固定化酶和溶液酶一样有底物抑制现象，固定化酶在6mol/L脲中处理6h活力趋向稳定，其活力为原有活力的46．75，用固定化酶处理啤酒，啤酒浊度下降了2－9．25倍，蛋白质含量下降了78．8％，冷藏120天，处理啤酒无冷混浊现象发生，同时啤酒原有风味和其它理化指标保持不变。     

SPSS正交设计提取林蛙油多糖

目的研究中性蛋白酶水解提取林蛙油多糖的最佳工艺参数。方法以料水比、中性蛋白酶浓度、水解时间和水解温度为考察因素,提取液中多糖含量为指标,通过SPSS软件设计L9(34)正交试验,并用SPSS进行数据处理。结果通过正交实验,得出中性蛋白酶提取林蛙油多糖工艺条件为料水比1∶100、加酶量为5%、水解时间1h、水解温度50℃。结论运用SPSS软件设计L9(34)正交表并进行数据处理,大大方便了实验结果处理,使实验方便科学,得出中性蛋白酶水解提取林蛙油多糖的工艺。     

CHEMICAL MODIFICATION OF NEUTRAL SUBTILOPEPTIDASE BY AROMATIC DIAZO COMPOUNDS

Effect of several aromatic diazo compounds on the activity of neutral protease of Bacillus subtilis var. amylosacchariticus (neutral subtilopeptidase) was investigated. Treatment of the enzyme with a 50-fold molar excess of p-nitrobenzene diazonium fluoroborate at pH 6.5 for 15 min reduced the caseinolytic activity, whereas it increased the activity toward synthetic substrate, Z-Gly-L-Leu. NH₂, to 180 to 250% of that of the native enzyme. Almost the same result was obtained by incubating the enzyme with a 50-fold molar excess of β-diazonaphthalene fluoroborate at pH 8.5–10.0 for 15 min. Spectrophotometric and amino acid analyses indicated that the azocoupling occurred exclusively at the tyrosyl residue(s) of the enzyme. Another diazo compound, 3-diazoquinoline fluoroborate, however, seemed to react with lysyl residue(s) of the enzyme, without significantly affecting the enzyme activity.     

乳源抗菌肽的分离纯化及部分性质的研究

建立酪蛋白酶解产生抗菌肽的最适条件，对酶解产物进行分离。建立抗菌肽活性测定方法，并对酪蛋白源抗菌肽的理化特性予以评价。本实验采用胰蛋白酶在不同时间条件下水解酪蛋白，经凝胶过滤和RP-HPLC分离纯化制备抗菌肽，酶解液及其分离物质对不同菌株的大肠杆菌和金黄色葡萄球菌的抗菌活性进行了测定。结果表明：2，3h胰蛋白酶的酪蛋白水解液均具有抗菌活性，且有良好的热稳定性。酪蛋白和胰蛋白酶本身无抗菌活性。2h水解液对大肠杆菌ATCC25922和金黄色葡萄球菌CMCC26801的最小抑菌浓度（Minimal Inhibitory Concentration, MIC））分别是1．20mg／ml和0．34mg／ml。2h水解液经SephadexG-15分离收集的5^#组分有抗菌活性，RP-HPLC检测有较单一肽吸收峰。说明酪蛋白经酶水解可以产生抗菌活性肽。     

THERMOLYSIN AND BACILLUS SUBTILIS NEUTRAL PROTEASE

A comparative study on thermolysin from Bacillus thermoproteolyticus and neutral protease from Bacillus subtilis involving far-and near-ultraviolet circular dichroism (CD) and immunological techniques is reported. These enzymes are homologous metalloendopeptidases, having similar size, kinetic behaviour, substrate specificity and susceptibility to inhibitors. The far-ultraviolet CD spectrum of each protein shows a minimum at 208 and a shoulder near 220 nm; differences in the extent of ellipticity, however, have been observed. Estimates of secondary structure obtained by quantitation of the far-ultraviolet CD spectra indicated a higher helicity of neutral protease relative to thermolysin. In the presence of ethylenediaminetetraacetic acid, which removes calcium and the functional zinc ion from the metalloenzymes, neutral protease is immediately denatured, whereas thermolysin maintains a globular structure, although thermolabile. On the other hand, the zinc-specific chelating agent tetraethylenepentamine does not have measurable effects on the conformation and conformational stability of either protein. Marked higher stability to temperature and guanidine hydrochloride were observed for thermolysin as compared with neutral protease, as indicated by monitoring conformational transitions with CD measurements at 220 nm. Antisera prepared in rabbits using thermolysin as immunogen do not cross-react with neutral protease, indicating differences of surface structure between the two proteins. On the basis and limitations of the techniques employed, it is proposed that the two sequentially and functionally homologous metalloendopeptidases may have similar conformations at specific regions (active and binding sites, at least) of the polypeptide chain essential for biological function, while some variability in the structure of other regions may be tolerated.     

Expression of Bacillus protease (Protease BYA) from Bacillus sp. Y in Bacillus subtilis and enhancement of its specific activity by site-directed mutagenesis-improvement in productivity of detergent enzyme-

An attempt was made to express protease BYA produced by an alkalophilic Bacillus sp. Y in Bacillus subtilis by gene engineering methods. The gene encoding protease BYA was cloned from Bacillus sp. Y, and expression vector pTA71 was constructed from the amylase promoter of Bacillus licheniformis, DNA fragments encoding the open reading frame of protease BYA, and pUB110. Protease BYA was secreted at an activity level of 5100 APU/ml in the common industrial culture medium of Bacillus subtilis transformed with pTA71. We then attempted to increase the specific activity of protease BYA by site-directed mutagenesis. Amino acid residue Ala29 next to catalytic Asp30 was replaced by one of three uncharged amino acid residues (Val29, Leu29, Ile29), and each mutant enzyme was expressed and isolated from the culture medium. Val29 mutant enzyme was secreted at an activity level of greater than 7000 APU/ml in culture medium, and its specific activity was 1.5-fold higher than that of the wild-type enzyme. Other mutant enzymes had specific activity similar to that of the original one and were less stabile than the wild-type enzyme. It can be thought that the substitution at amino acid residue 29 affects the level of activity and stability of protease BYA.     

Cytotoxic potential of industrial strains of Bacillus sp

The cytotoxic potential of selected strains of Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis, used in the production of industrial enzyme products, has been assessed. Cytotoxicity was determined in Chinese hamster ovary (CHO-K1) cells by measuring total cellular metabolic activity using the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Initially the MTT assay was validated against toxigenic strains of Bacillus cereus, to define the exact criteria for a toxigenic versus a nontoxigenic response. The assay proved sensitive to culture broths of both a diarrheagenic strain and an emetic strain of B. cereus. The enzyme-producing strains tested were nontoxic to CHO-K1 cells. Additionally it was demonstrated that our industrial strains did not react with antibodies against B. cereus enterotoxins by use of commercial antibody-based kits from Oxoid and Tecra. A short survey of the literature concerning the toxigenic potential of species within the subtilis group is included, as is a database search of known B. cereus enterotoxins against B. subtilis and B. licheniformis DNA sequences.     

枯草芽孢杆菌guaB基因克隆及其在大肠杆菌中的表达

克隆枯草芽孢杆菌guaB基因,构建穿梭表达载体,使其在大肠杆菌中获得表达,为构建鸟苷高产工程菌奠定基础。从枯草芽孢杆菌JSIM-G-518基因组扩增出guaB基因,将其连接到pMD19-T上,得到重组质粒pMD-guaB,进行测序和Blast比对分析。该重组质粒经PstI、BamHI双酶切,获得guaB片段连接至pHP13载体上,构建穿梭表达载体pHP13-guaB,并采用双酶切电泳进行鉴定。将该载体转化入大肠杆菌BL21中,通过SDS-PAGE电泳检测guaB基因的表达效果。克隆的guaB基因长度为1 510 bp,与GenBank登录的枯草芽孢杆菌同源性为99%,编码500个氨基酸,确认为控制IMP脱氢酶的基因。穿梭载体pHP13-guaB经双酶切后产生1 510 bp和4.9 kb两个期望的目的条带。SDS-PAGE结果显示表达产物相对分子质量的大小为52.9 k,与预期的IMP脱氢酶分子量一致,且该目的蛋白的表达效果比对照组更明显。论文成功实现了枯草芽孢杆菌guaB基因在大肠杆菌中的表达。     

垂盆草总黄酮的酶法提取及其抑菌活性

目的研究垂盆草总黄酮的酶法提取工艺及其抑菌作用。方法用酶解和煎煮相结合的方法,以总黄酮得率为指标,通过正交实验优选出最佳提取工艺,用平板打孔法考察垂盆草总黄酮的抑菌作用。结果垂盆草总黄酮的最佳酶解工艺条件是酶解温度50℃,酶解pH 6.0,酶用量0.6%,酶解时间1.5 h,其中酶解温度是显著影响因素。总黄酮得率可达3.471%。垂盆草总黄酮提取液对大肠杆菌、金黄色葡萄球菌、枯草杆菌和产气杆菌都有不同程度的抑菌作用,对四联球菌作用不明显。结论该实验得到的垂盆草总黄酮的提取工艺合理可行,提取率高,提取得到的总黄酮具有一定的抑菌作用。     

芽孢杆菌BI1的鉴定及其抑菌性研究

目的 对本实验室分离得到芽孢杆菌B11进行鉴定.方法 将该菌株的16S rDNA基因序列与GeneBank中已鉴定的16S rDNA序列对比,并结合生理生化实验综合分析.结果 菌株B11与枯草芽孢杆菌亲缘关系最为接近,16S rDNA基因相似性达到99.6%,生理生化实验结果与标准枯草芽孢杆菌一致.结论 将芽孢杆菌B11鉴定为枯草芽孢杆菌.     

戊二醛溶液的稳定性考察

目的:考察戊二醛溶液持续使用天数及次数与稳定性的关系。方法:用试管稀释法确定中性与碱性戊二醛的最低杀菌浓度,并采用羟胺缩合紫外分光光度法监测两者在持续使用过程中及使用次数不同情况下的含量变化。结果:碱性戊二醛对大肠杆菌、金黄色葡萄球菌的MBC大于中性戊二醛,对枯草芽孢杆菌则反之;两者含量均随放置时间不断下降,下降幅度碱性戊二醛大于中性戊二醛,而使用次数对其影响不大。结论:中性戊二醛对大肠杆菌、金黄色葡萄球菌杀菌力强且稳定性高,碱性戊二醛对枯草芽孢杆菌杀菌力较强但稳定性低。