角蛋白的微生物降解与利用

本文对角蛋白、角蛋白酶、角蛋白降解菌的研究和应用状况作了系统的综述，现代化的家禽养殖产生大量角蛋白废物（如羽毛和毛发），若加以合理利用，它们是一种新的蛋白质来源。多种角蛋白降解菌和角蛋白酶已被分离并得到深入研究。在强调可持续发展的今天，角蛋白酶的研发和角蛋白废的利用不失为一个经济有效的环保举措。     

蹄甲角蛋白酶高产菌株的紫外诱变及酶学性质研究

为了得到蹄甲角蛋白酶高产菌株,本试验对1株产角蛋白酶的黄杆菌(Chryseobacteriumsp.)N5菌株进行紫外诱变,筛选得到高产突变株U3-22,利用考马斯亮蓝法测定U3-22菌株的角蛋白酶发酵活力达69.9U/mL,比出发菌株的25.4U/mL提高了2.75倍。该角蛋白酶最适反应温度是70℃,最适pH是7.5;K+、Mg2+、Na2SO3对该角蛋白酶有较明显的促进作用,而Mn2+、Zn2+的抑制作用较为明显;突变株U3-22产生的角蛋白酶对蹄甲粉具有很强的分解能力,且具有较好的热稳定性和pH稳定性。结果表明,U3-22产生的角蛋白酶是一种新型的耐高温、耐酸碱角蛋白酶,在动物蹄甲、羽毛等废弃资源的利用中有巨大的应用前景。     

嗜麦芽窄食单胞菌DHHJ角蛋白酶的理化性质

通过羽毛粉的酶解实验，研究了嗜麦芽窄食单胞菌（Stenotrophomonas maltophilia）DHHJ突变菌株的粗酶液的理化性质。结果表明，酶水解羽毛的最适条件为pH7．8、温度50℃，在pH7．0～8．0、60℃以下酶活较稳定。Ca^2＋、Ba^2＋、Cu^2＋、Na^＋、K^＋和Mg^2＋对粗酶活力有促进作用，而Hg^2＋、Cd^2＋、Pb^2＋、Zn^2＋和苯甲基磺酰氟（PMSF）则对粗酶活力有抑制作用。对粗酶液进行了十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS—PAGE），根据测定结果认为该角蛋白酶可能为复合酶，由两个亚基构成，分子量分别为141kD和119kD，并认为这是一种新型角蛋白酶。     

羽毛粉部分替代鱼粉对异育银鲫生长及抗氧化能力的影响

【目的】探讨羽毛粉部分替代鱼粉外加角蛋白酶DP100(DP100)对异育银鲫生长及抗氧化能力的影响。【方法】配制7组饲料,Ⅰ组(对照组)以鱼粉为蛋白源(蛋白水平为317.9 g/kg),Ⅱ～Ⅶ组以羽毛粉蛋白替代10%的鱼粉蛋白,Ⅲ～Ⅶ组添加Ⅰ组相对短缺的4种必需氨基酸并分别添加0,100,200,300和400 mg/kg的DP100。用7组饲料分别饲喂异育银鲫83 d,试验结束后根据异育银鲫初始体质量、终末体质量(FBW)以及摄食量计算体质量增加率(BWG)、特定生长率(SGR)、饲料效率(FER)、蛋白质效率(PER),并解剖取肝胰脏和肠道,测定其消化酶(蛋白酶、脂肪酶、淀粉酶)活力及抗氧化指标(超氧化物岐化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活力及丙二醛(MDA)含量)。【结果】Ⅱ和Ⅲ组异育银鲫FBW、BWG、SGR、FER、PER及肝胰脏和肠道蛋白酶、脂肪酶、淀粉酶、SOD、CAT、GSH-Px活力均显著低于Ⅰ组(P0.05),MDA含量显著高于Ⅰ组(P0.05);在添加DP100的各组中,Ⅳ组肠道SOD活力和肝胰脏CAT活力均与Ⅰ组无显著性差异,MDA含量显著高于Ⅰ组(P0.05),其余指标均显著低于Ⅰ组(P0.05),除Ⅶ组肠道蛋白酶活力显著低于Ⅰ组(P0.05)外,Ⅴ、Ⅵ和Ⅶ组各项指标均与Ⅰ组无显著性差异。【结论】羽毛粉替代10%鱼粉外加200～400 mg/kg DP100对异育银鲫生长性能、饲料利用和机体抗氧化能力无不良影响。     

产角蛋白酶毕赤酵母工程菌的构建及酶学性质

将Brevibacillus brevis US575角蛋白酶基因kerUS进行密码子优化,并在毕赤酵母GS115中表达,以提高角蛋白酶产量。结果表明:优化的基因可在毕赤酵母中成功表达,重组角蛋白酶的分子质量约30 ku,最佳反应条件为pH 9.5和60℃,1 mmol/L的Mn~(2+)、Ba~(2+)对角蛋白酶活性有促进作用。此外,重组角蛋白酶易降解干酪素和β角蛋白(羽毛粉),酶促反应最大速度v_(max)=0.019 g/(L·min),K_m=4.64 g/L,比活力=440 U/mg。研究获得的产角蛋白酶重组毕赤酵母具有较好的应用前景。     

羽毛角蛋白降解的研究进展

羽毛角蛋白是一种硬性蛋白,在动物饲料、复合材料、食品包装和医药制品等领域展现出良好应用前景。但因羽毛角蛋白难溶解和降解的特殊性,一般方法很难获得可溶性角蛋白,酶法相对于传统物理和化学降解法具有反应条件温和、环境污染小和降解产物稳定等优点,是具有很高实用价值的羽毛降解途径。文中重点介绍角蛋白酶对羽毛的降解作用,并概述物理、化学方法的降解原理以及角蛋白的综合利用。     

Isolation and Identification of A Keratinase-producing Strain of Pseudomonas aeruginosa B1-2

A new feather-degrading bacterium was isolated from soil samples colected from pile-up feather places in this study.The transparent circle was produced after the strain was cultured on the milk medium for 24 h,the colony shape of the strain was irregular,rough surface,edge blur,and could produce green fluorescent pigment.It was gram negative,short rod,nospore and capsule and had a flagellum scanning by electron microscope. Biochemical test showed that the isolate could hydrolyze milk and gelatin,utilize citrate with keratinase activity and nitrate reduction reaction positive.Phylogenetic tree analysis showed that the isolate was most closely related to Pseudomonas aeruginosa strain (GenBank accession No.:BAMA01000316).Combined with the above results,the isolated strain was identified as Pseudomonas aeruginosa B1-2. When it was inoculated on basal medium containing 1% feather as sole carbon and nitrogen source,the highest level of keratinase activity was 60.3 U/mL after 48 h while the degradation rate of feather was 85.7%. This result indicated that the newly isolated Pseudomonas aeruginosa B1-2 could be useful in management of farm wastes.     

Beneficial Effects of Versazyme,a Keratinase Feed Additive,on Body Weight,Feed Conversion,and Breast Yield of Broiler Chickens

Versazyme (VZ) is a keratinase-based feed additive. Broiler diets were formulated with low (95%), medium (100%), or high (105%) CP and amino acid levels relative to US Poultry Industry Reporting Service averages (2003). In each diet, amino acid minimums were maintained relative to the percentage of CP. The protein levels were as follows: starter—21, 22, 23%; grower—19, 20, 21%; finisher—17, 18, 19%; and withdrawal—16, 17, 18%. Mixed sex broilers 0 to 48 d of age were fed the 3 protein levels with and without VZ (0.10%) in a 3 × 2 factorial experiment. Statistical main effects indicated 48-d BW was greater on the high protein diets (2.42 kg) compared with low protein (2.36 kg), with the medium protein diet being intermediate (2.40 kg). Overall, VZ significantly (P < 0.01) improved 48-d BW 2.54% (from 2.36 to 2.42 kg, P < 0.01), feed conversion ratio 1.62% (from 1.912 to 1.881, P < 0.01), and breast meat yield 1.68% (from 31.57 to 32.10%, P < 0.01). The improvements in BW and feed conversion ratio by VZ were greatest with the low protein diets. However, VZ produced the greatest improvement in breast meat yield at the medium and high protein levels. This enzyme feed additive apparently improves the utilization of amino acids by broilers fed diets formulated to commercial specifications.     

一株产角蛋白酶的铜绿假单胞菌的分离鉴定

本研究从鸡场长期堆放羽毛的土壤中筛选到一株降解羽毛能力强的菌株。该菌株在牛奶平板上培养24 h后产生透明降解圈,形状不规则,表面粗糙,边缘模糊,产生绿色荧光色素;革兰氏染色阴性,短杆状,无芽孢,有鞭毛,无荚膜。生化试验显示,该分离株能够水解牛奶、明胶,具有角蛋白酶活性,能利用柠檬酸盐,硝酸还原反应阳性。系统进化树分析显示,分离株与铜绿假单胞菌模式菌株（GenBank登录号：BAMA01000316）亲缘关系最近,综合以上结果,最终确定分离株为铜绿假单胞菌,并正式命名为Pesudomonas aeruginosa B1-2。将该分离株在以1%羽毛为唯一碳氮源的基础培养基中发酵培养,48 h后角蛋白酶活性最高达到60.3 U/mL,对羽毛降解率为85.7%。本研究中分离获得的Pesudomonas aeruginosa B1-2可用于畜禽废弃物的处理。     

Evaluation of Aqueous Biphasic Electrophoresis System Based on Halide-Free Ionic Liquids for Direct Recovery of Keratinase

Aqueous biphasic electrophoresis system (ABES) incorporates electric fields into the biphasic system to separate the target biomolecules from crude feedstock. Ionic liquid (IL) is regarded as an excellent candidate as the phase-forming components for ABES because of the great electrical conductivity, which can promote the electromigration of biomolecules in ABES, and thereby enhances the separation efficiency of the target biomolecules from crude feedstock. The application of electric fields to the conventional biphasic system expedites the phase settling time of the biphasic system, which eases the subsequent scaling-up steps and reduces the overall processing time of the recovery process. Alkyl sulphate-based IL is a green and economical halide-free surfactant when compared to the other halide-containing IL. The feasibility of halide-free IL-based ABES to recover Kytococcus sedentarius TWHK01 keratinase was studied. Optimum partition coefficient (K_e = 7.53 ± 0.35) and yield (Y_T = 80.36% ± 0.71) were recorded with IL-ABES comprised of 15.0% (w/w) [EMIM][ESO₄], 20.0% (w/w) sodium carbonate and 15% (w/w) crude feedstock. Selectivity (S) of 5.75 ± 0.27 was obtained with the IL-ABES operated at operation time of 5 min with 10 V voltage supplied. Halide-free IL is proven to be a potential phase-forming component of IL-ABES for large-scale recovery of keratinase.