【作者】 黄志强；

【导师】 谢联辉；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 本研究从福建海域的海水、海泥及海藻样品中分离到三株产碱性蛋白酶的海洋细菌菌株3B、6CW、15E，采用16S rDNA分子生物学鉴定结合细菌的常规鉴定方法，确认分离到的三个产蛋白酶的海洋细菌菌株分别是荧光假单胞菌（Pseudomonas fluorescens），粘质沙雷菌（Serratia marcescens），氧化短杆菌（Brevibacterium oxydans）。其中，海洋氧化短杆菌（Brevibacterium oxydans）产碱性蛋白酶为首次报道。 对氧化短杆菌15E菌株产碱性蛋白酶的发酵条件进行研究，结果表明：在2％接种量、培养基的初始pH为9.0、装液量在20％左右、培养温度为28℃、摇床转速为200r／min的条件下培养40h时，菌体生长最适且总酶活达到最大；菌株在添加有0.5％牛肉膏、0.1％葡萄糖的酪蛋白培养基中生长和产酶均达到最佳；人工海水中的K⁺、Ca²⁺和Mg²⁺浓度较为适合15E菌株生长和产酶，其中K⁺对菌株产酶起关键作用。 菌株15E所分泌的碱性蛋白酶的分子量为49.0kDa，等电点为9.3，米氏常数Km为5.6×10^-3g／mL。该酶的最适作用温度为60℃，最适pH值为7.0-9.0；Ca²⁺、Mg²⁺、K⁺对酶有激活作用。酶对一些变性剂如SDS、尿素、盐酸胍具有一定的耐受性；PMSF和AEBSF能抑制该蛋白酶，表明该酶属于丝氨酸蛋白酶；EDTA对该蛋白酶有抑制作用，说明该蛋白可能含有金属离子。 氧化短杆菌（Brevibacterium oxydans）菌株15E碱性蛋白酶apr基因克隆结果表明，该DNA片段长度为1357nt，最大开放阅读框编码447个氨基酸。从第122个氨基酸到第144个氨基酸这段序列可能是一个跨膜区。但是，获得的序列经分析并未存在特征明显的信号肽序列。利用系统发育树分析，所克隆的序列与褐色高温单胞菌碱性蛋白酶前导序列和铜绿假单胞菌碱性蛋白酶基因处于同一个分支，说明该序列为编码碱性蛋白酶的基因。同源性分析结果显示，该蛋白可能是一个结合蛋白酶类，存在一个活性中心。更多还原

【Abstract】 Three alkaline protease-produced marine bacterial strains, named as 3B, 6CW and 15E, were isolated form samples of sea-water,sea-mud and seeweed. These isolates were characterized and identified as species of Pseudomonas, Microbacterium, and Brevibacterium respectively, based on their physiological and biochemical characteristics and analysis of 16S rDNA sequence.And, marine bacterium Brevibacterium oxydans secreting alkaline protease is firstly reported.Fermentation conditions of the alkaline protease secreting from Brevibacterium oxydans 15E were researched. In the condition that initial pH of culture medium was 9.0, culture-broth-quantum was about 20%, inoculum was 2%, speed of shake-culture was 200 r/min and culture time was 40 hours, the strain 15E grew well and total protease activity reached its peak. The casein medium adding 0.5% beef extract and 0.1% glucose was suitable for the growth of the organism and the production of the protease. The concentration of K⁺, Ca²⁺ and Mg²⁺ in the artificial-seawater was suitable for the growth and protease synthesis of strain 15E, while K⁺ was vital for the protease production.Strain 15E could secrete alkaline protease, whose molecular weight was 49.0 kDa, isoelectric point was 9.3 and Km value was 5.6× 10^-3 g/mL. The optimum condition for the protease activity was 60℃ and pH ranging from 7.0 to 9.0. Ions Ca²⁺,Mg²⁺ and K⁺ promoted the activity of the protease.The protease was tolerable to some denaturing agents such as SDS, Urea, Guanidine HC1. PMSF and AEBSF could greatly inhibit protease activity, which indicated the alkaline protease belonged to the type of serine protease; the enzyme could also be inhibited by EDTA, which showed that some metal ions played a role on the protein for catalytic activity.The result of cloning alkaline protease gene apr showed that the length of sequence in nucleotides is 1 357 nt, and the max ORF encoded 447 amino acid. Amino acid sequences of transmembrane were from 122th to 144th amino acid, and it was probably a precursor更多还原