Bovine metalloprotease characterization and in vitro connective tissue degradation

Metalloproteases that selectively hydrolyze connective tissue proteins may tenderize meat without creating texture problems associated with myofibrillar protein degradation. Our objective was to characterize the activity of bovine placental proteases to determine whether they can improve meat tenderness through disruption of the connective tissue matrix. Enzymes were extracted, crudely purified, and proteolytic activity was assessed against gelatin and collagen under varying pH and temperature conditions using both SDS-PAGE and zymography. Gelatin zymography revealed proteolysis between 57 and 63 kDa, with decreased activity as buffer pH decreased from pH 7.4 to 5.4 (37 degrees C). Proteolytic activity was pronounced at 37 degrees C, moderate at 25 degrees C, and absent at 4 degrees C following 48-h incubation (pH 7.4). Placental enzymes were metalloproteases inhibited by excess EDTA. Maximum proteolysis was achieved in the presence of Ca2+, with or without Mg2+ and Zn2+. Absence of Ca2+ decreased proteolytic activity. Complete degradation of both the 125- and 120-kDa proteins of the alpha-chains of gelatin was achieved following enzyme incubation for 6 h at 37 degrees C or 24 h at 25 degrees C. No degradation was observed following enzyme incubation with native Type I collagen. Given the marked decrease in enzyme activity at pH 5.4 and 4 degrees C (standard industry conditions), bovine placental metalloproteases would not be expected to contribute to connective tissue degradation or improve meat tenderness.     

鸡毒支原体磷酸酶PrpC活性检测

HPr激酶/磷酸酶(PrkC/PrpC)系统在细菌和支原体中高度保守,不仅参与糖酵解酶类的磷酸化/去磷酸化,也与毒力、生物被膜等生命活动相关。克隆并突变获得编码鸡毒支原体磷酸酶PrpC的ptc1基因,经原核表达纯化后,利用底物PNPP体外检测其酶活性,并对影响该酶活性的pH、金属离子、温度等影响因子进行分析。结果表明,Ptc1蛋白具有磷酸酶活性,Mn2+为该酶辅因子,最适离子浓度为2mol/L,最适pH为8.5,最适温度为37℃。相同浓度的Li+、Na+、K+、Mg2+、Ca2+、Ba2+、Al 3+、Hg2+等金属离子均对表达产物具有不同程度的抑制活性。初步建立了PrpC功能检测体系,为进一步深入研究PrkC/PrpC调节系统奠定基础。     

大理弥渡热泉耐热脂肪酶产生菌的筛选及其酶活性研究

从云南大理市弥渡县石夹泉热泉的43℃底泥中筛选到1株高温脂肪酶的高产菌,进行显微形态及生理生化特征、16S rRNA基因序列分析,将其初步鉴定为不动杆菌属(Acinetobacter sp.)的一株菌,命名为Acinetobacter sp.Lip-43。对其生长条件及酶学性质进行了研究,结果表明:该菌株耐高温性较好,菌株在55℃仍能生长,菌株最适生长温度为37℃。所产脂肪酶最适酶活温度为45℃,最适反应pH为5.0。在37℃以下,能保持良好的稳定性,Zn^2+和Ca^2+对该酶有一定的抑制作用,Mn^2+、K^+、Mg^2+、Fe^2+、Cu^2+均对该酶活力起到促进作用,其中Mn2+的促进效果最为明显。     

家蚕5龄幼虫体内共轭酶和二氢叶酸还原酶的基本性质

共轭酶 (EC 3 4 2 2 12 )和二氢叶酸还原酶 (EC 1 5 1 3)是生物体内重要的叶酸代谢酶。以家蚕幼虫为材料分析了这两种酶的基本性质。 5龄幼虫体液共轭酶反应的最适pH为 7 8,最适温度为 5 0℃ ;反应体系中添加巯基乙醇、K+ 或Mg2 + 可显著提高酶活性 ,而对羟高汞苯磺酸、Co2 + 、Zn2 + 、Fe2 + 等对酶活性具有明显的抑制作用 ;以叶酸五谷氨酸为基质的水解产物为叶酸 ;另以叶酸三谷氨酸为底物 ,采用 33mmol/LHepse Ches缓冲液 (pH 7 8)在 37℃的反应条件下 ,求得Km 为 9 6 μmol/L。幼虫脂肪体等组织中二氢叶酸还原酶以NADPH为供氢体 ;在pH 4 5～7 5的范围内具有较强活性 ,最适反应温度 35℃ ;巯基乙醇、胍 ,以及高浓度的 1价金属离子 (K+ 、Na+ 等 )对酶具有活性化作用 ,对氯高汞苯甲酸、甲酰胺、Co2 + 等具有强烈的抑制作用 ;采用 35mmol/L柠檬酸 磷酸钠缓冲液 (pH 5 .0 ,10g/L抗坏血酸 )在 30℃的反应条件下 ,求得Km 值为 2 7μmol/L。家蚕体液可作为共轭酶的新酶源而加以利用。     

产α-半乳糖苷酶菌株的筛选、鉴定及其酶学特性的研究

利用筛选培养基从不同豆制品作坊附近的土样中筛选出一株产α-半乳糖苷酶的菌株D-1,对其进行分子鉴定及所产的α-半乳糖苷酶进行酶学特性研究。研究表明,菌株D-1为Aspergillus niger,此菌株所产α-半乳糖苷酶的最适反应温度是55℃,在60℃以下热稳定性较好;最适反应pH值为5.0,在pH值3.0～5.5范围内稳定性较好,相对酶活>64.1%;Mg2+、Na+、Pb2+、K+、Mn2+、Co2+、Al3+对α-半乳糖苷酶均有不同程度的抑制作用,其中Cu2+和Fe3+的抑制作用较为明显,而Ca2+、EDTA(乙二胺四乙酸)、Zn2+对α-半乳糖苷酶有一定的促进作用。     

Glycerolipid biosynthesis in porcine adipose tissue in vitro. II. Synthesis by various types of cellular preparations

Glycerolipid biosynthesis by porcine adipose tissue homogenates did not yield the 90+% triacylglycerol observed in situ. Consequently, we compared intact tissue slices and various subcellular fractions to characterize the usefulness of such systems to assess glycerolipid biosynthesis in vitro. Glycerolipid biosynthesis by porcine adipose tissue homogenates was measured in vitro using either [14C]-fatty acid or [14C]-glycerol-3-phosphate (G3P) as a radiolabelled substrate. Removal of residual 14C-labelled fatty acid from lipid extracts was difficult. Because G3P is soluble in water, residual [14C]-G3P separated easily from the glycerolipid-containing organic phase and, thus, was the preferred radiolabelled substrate. With tissue slices, glycerol and G3P were minimally incorporated into lipid so that [14C]-fatty acid was the preferred radiolabelled tracer. A washing procedure followed by thin layer chromatography was devised to separate residual [14C]-fatty acid from glycerolipids, including phospholipids. Fatty acid esterification into glycerolipids in tissue slices yielded about 4% phospholipids, whereas with homogenates, esterification yielded up to 50% phospholipids. Comparison of several subcellular fractions indicated that microsomes contained most of the glycerolipid biosynthetic activity when activity was expressed on a protein basis. However, when activities were expressed on a tissue wet weight basis, the 700 x g infranate and the 10,000 x g supernate had about equal activity that was far greater than the microsomes. The 700 x g infranate was the preferred enzyme preparation for assay of the entrance of G3P into the pathway as well as the capacity to synthesize triacylglycerol. Several methods of freezing and storing tissue or 700 x g infranates were not acceptable. Freezing of the 700 x g infranate in liquid N2 with storage at -80 degrees C may be an acceptable procedure.     

饲用α-半乳糖苷酶酶活稳定性的研究

在酶制剂生产加工和储存过程中,酶活稳定性是酶应用过程中最常遇到的实际问题。本文研究不同载体、高温高湿、金属离子、保存初始pH值、保护剂浓度和存储时间对体外α-半乳糖苷酶稳定性的影响。试验结果表明:①无机载体碳酸钙、元明粉和滑石粉对α-半乳糖苷酶活性影响很大,收率均为0%;有机载体中玉米皮粉效果最好,收率高达90.83%;其次是淀粉,收率为61.11%;稻壳粉收率最低,仅为44.72%。②湿度为17%,温度小于85℃,酶活损失率小于10%,说明该酶有较强的耐温能力,温度为90℃,酶活损失率达到16.2%,随着温度上升,酶活损失率开始增加,不利于酶活的保存。③Cu2+对该酶有抑制作用3,7℃恒温水浴4 h后酶活存留率仅为74%,Mn2+、Zn2+、Ca2+、Na+、K+和Mg2+对α-半乳糖苷酶有几近相同的保护作用,酶活存留率均大于92%。④该酶最适保存pH值为5.3;pH值为4.4时,酶活损失率达到30.1%,不利于酶的保存;pH值为5.6时,酶活损失率为3.77%,pH值在4.7~5.6之间,酶活损失率小于10%,有利于酶的保存。总之,过酸和过碱都不利于酶的保存。⑤1%NaCl、1%甘露醇、5%和7%的山梨醇都有杂菌产生,不利于酶的保存3;%~5%的NaCl酶活损失小于10%1,%和5%~7%的甘油酶活损失率小于5%,都有利于酶活保存。     

Effects of Pasteurella haemolytica leukotoxin on cultured bovine lymphoma cells

Leukotoxin activity from culture supernatants of Pasteurella haemolytica serotype 1 in logarithmic growth phase caused rapid (less than 5 min) release of intracellular K+, uptake of extracellular Ca2+, and swelling of cultured bovine lymphoma cells (BL3 cells). Release of 51CrO4(2-) and lactate dehydrogenase (LDH) from BL3 cells began after 15 minutes of incubation with leukotoxin at 37 C and was completed between 60 and 120 minutes of incubation. In addition, leukotoxin exposure of BL3 cells resulted in cell aggregation and adherence to glass surfaces. Scanning electron microscopy indicated that after 10 minutes of leukotoxin exposure, BL3 cells increased in size, and large membrane defects developed between 20 and 60 minutes of exposure. The rate of release of LDH from leukotoxin-exposed BL3 cells was proportional to the amount of leukotoxin added. At high cell concentrations, the activity of LDH released at completion was directly proportional to the amount of leukotoxin added. Leukotoxin-induced release of LDH required a divalent cation, whereas K+ release and cell swelling did not. The addition of Ca2+, Mn2+, and Ba2+ resulted in increased leukotoxin-induced release of LDH. Divalent cation concentrations of 0.5 to 2.5 mM resulted in 50% of maximal stimulation. Ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid blocked increased release of LDH caused by Ca2+ addition, but had no effect on K+ release or cell swelling. Leukotoxin action on BL3 cells (K+ release, cell swelling, Ca2+ uptake, and release of LDH) was prevented by incubation at 4 C.     

Comparisons of four methods for quantification of lysosomal cysteine proteinase activities

Four methods were compared to optimize the measurement of the activities of cathepsin B and cathepsin L in porcine skeletal muscle. These methods were: Method A (lysosomal enriched fraction obtained by differential centrifugation), Method B (muscle extract in the absence of detergent), Method C (muscle extract in the presence of detergent) and Method D (the same as method C, but passed through a S-carboxymethylated-papain-Sepharose affinity column). Results indicated that, of the methods tested, Method D yielded greater cathepsin B and consistently greater cathepsin B + L activities per gram of muscle. Hence, Method D is the method of choice for quantification of these enzyme activities. Studies indicated that for cathepsin B with Z-Arg-Arg-NMec as substrate, Km and Vmax values were .416 mM and 4,405 pmol.min-1.mg protein-1, respectively. The Km and Vmax for cathepsins B + L were .132 mM and 9,346 pmol.min-1.mg protein-1, respectively. The relationship between enzyme activity and incubation time was linear for the incubation times studied (up to 60 min). Also, the relationship between enzyme activities and amount of protein in the assay was linear at the concentrations studied (up to 20 micrograms protein). The same preparations were assayed by conditions commonly used by many investigators (.005 mM substrate, approximately 75 micrograms protein, 30 min at 37 degrees C) and by conditions established in this study (1.0 mM substrate, 10 micrograms protein and 15 min at 37 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Some properties of beta-D-galactosidase from the adult filarial nematode Setaria digitata

beta-D-galactosidase (beta-D-galactoside galactohydrolase, E.C. 3.2.1.23) activity was localised in the digestive tract of Setaria digitata. The enzyme extract shows maximum activity in the pH range between 3.5 and 5.0 and at 45 degrees C. The enzyme shows the Km value of 3.636 mM for the substrate 6-bromo-2-naphthyl beta-D-galactoside and Vmax of 28.57 nmol 6-bromo-2-naphthol liberated mg-1 protein min-1. Activation/inhibition of the enzyme by various ions, medicinal plants and drugs has been studied. Polyacrylamide gel electrophoresis revealed that the enzyme exists as single form. The medicinal plants and the drug filarin effectively inhibit the enzyme. The significance of these results are discussed in relation to chemotherapy.     

Comparison of methods for assay of fecal proteolytic activity

Fecal proteolytic activity (FPA) in ten normal dogs was readily detected either calorimetrically using azocasein substrate or by radial enzyme diffusion into agar gels containing casein substrate. Daily FPA ranged from 17 to 207 azocasein units/g (ACUIG) or 4 to 18 mm of casein hydrolysis, while mean 3-day FPA ranged from 58 to 10 1 ACUIG or 7 to 15 mm. Studies of proteolytic activity remaining after treatment of fecal extracts with a specific trypsin inhibitor indicated that trypsin accounted for 0% to 71% of proteolytic activity. Proteolytic activity decreased steadily in fecal specimens stored at room temperature or above, but there was only slight loss in activity during storage for up to 5 days at 4 degrees C. Proteolytic activity was unaffected by repeated freezing and thawing and samples could be stored for long periods at -2 degrees C without noticeable loss of activity. It is concluded that assays of FPA using either azoprotein substrate or radial enzyme diffusion into agar gels containing casein substrate allow evaluation of exocrine pancreatic function in dogs, provided that several samples are tested. These methods are suitable for application in a variety of species.     

一株高产内切纤维素酶贝莱斯芽孢杆菌的产酶条件优化及酶学性质分析

试验旨在对一株杜仲树皮内生菌贝莱斯芽孢杆菌(Bacillus velezensis)的内切纤维素酶(CMCase)进行产酶条件优化及酶学性质分析,为探究贝莱斯芽孢杆菌CMCase的相关特性提供参考依据。采用单因子试验设计,分别优化pH、接种量、温度、时间、碳源和氮源等发酵条件,并探讨温度、pH、底物专一性、金属离子及表面活性剂对CMCase的影响。结果表明,以玉米秸秆为碳源(30 g/L)、豆粕为氮源(30 g/L)、3%接菌量、pH 7.0的条件下37℃培养48 h,贝莱斯芽孢杆菌157的CMCase最高可达(5.14±0.18) U/mL。经CMCase酶学性质分析发现,贝莱斯芽孢杆菌157的最适酶反应温度为60℃,最适pH为5.0;温度稳定性试验发现,在40和50℃时,相对剩余酶活力均高于80%,随着温度的升高,酶活力逐渐降低,当温度高于55℃,相对剩余酶活力约为50%;pH稳定性试验发现,在pH 5.0～10.0之间耐受性良好,相对剩余酶活力均高于90%,随作用时间的延长,相对剩余酶活力变化不大。贝莱斯芽孢杆菌157 CMCase属于典型内切型纤维素酶。Na^+、Mg²⁺、Ca²⁺可促进贝莱斯芽孢杆菌157 CMCase酶活力,而Co²⁺、Hg²⁺、Fe²⁺、Cu²⁺和SDS抑制CMCase酶活力;表面活性剂Tween-80、Tween-20、Triton X-100对贝莱斯芽孢杆菌157 CMCase无影响。本试验通过对贝莱斯芽孢杆菌157进行产酶条件优化及CMCase酶学性质分析发现,贝莱斯芽孢杆菌157 CMCase具有产酶量高,耐受pH范围广的特点,在饲料添加剂、洗涤剂和造纸领域中具有一定的应用价值。     

Ca2+ ATPase in Dutch warmblood foals compared with Na+, K+ ATPase: intermuscular differences and the effect of exercise

We studied the effects of exercise without or with a subsequent period on pasture on Ca2+ ATPase concentration in foal skeletal muscle, and compared the results with those previously reported on Na+, K+ ATPase. Ca2+ ATPase was measured in homogenates as Ca2+-dependent steady-state phosphorylation from [gamma-32P]ATP. From day 7 after birth, 24 foals were divided into three groups: (i) staying in a box stall (Box); (ii) staying in a box stall with an exercise programme of an increasing number of sprints per day (Exercise); and (iii) staying on pasture (Pasture). Half of the foals (12 with four in each treatment group) were killed after 5 months. The remaining foals stayed on pasture until 11 months. In the 5-month Pasture group, Ca2+ ATPase concentration was 29.4 +/- 4.3 nmol/g wet weight (wt) (n = 4) in gluteus medius muscle, 25.2 +/- 3.3 nmol/g wet wt (n = 4) in semitendinosus muscle (both mixed fibre type), and 4.1 +/- 1.7 nmol/g wet wt (n = 3) in the slow masseter muscle. These values were not altered by exercise or by box rest. This was in contrast to the Na+, K+ ATPase concentration which was not different between the three muscles, but showed a 20% rise in gluteus medius and semitendinosus muscle after exercise. In the period from 5 to 11 months on pasture, there was no change in Ca2+ ATPase in any group. In conclusion, the Ca2+ ATPase concentration in foal muscle is around 6-fold higher in mixed fibres than in slow fibres. Furthermore, the enzyme is not up- or down-regulated by sprint exercise or subsequent rest.     

Properties of partially purified goat kidney beta-D-mannosidase

Lysosomal beta-D-mannosidase (EC 3.2.1.25) was purified 6900-fold from normal goat kidney by serial Concanavalin A-Sepharose 4B and Red A dye ligand affinity chromatography, followed by anion exchange and molecular sieve high performance liquid chromatography. The relative molecular mass of the enzyme was estimated by molecular sieving to be 79,000 +/- 3000. The apparent Km for the synthetic substrate, 4-methylumbelliferyl-beta-D-mannopyranoside, was 2.3-2.8 mM and the sharp, unimodal pH optimum was 5.5. Enzyme activity was inhibited by Hg2+, Zn2+, Ag+, Co2+ and the thiol reactive agent N-ethylmaleimide. The mannose derivatives p-nitrophenyl-beta-D- thiomannopyranoside and p-aminophenyl-beta-D-thiomannopyranoside inhibited enzyme activity and may be of use as immobilized ligands in future attempts to purify beta-D-mannosidase by specific affinity chromatography.     

Modulation by sphingosine of phosphorylation of substrate proteins by protein kinase C in nuclei from cow mammary gland

Protein kinase C (PKC) is an enzyme activated by diacylglycerols such as 1-oleoyl-2-acetyl-sn-glycerol (OAG), phospholipids (in particular phosphatidylserine; PS) and Ca2+, which regulate a wide variety of intracellular functions by phosphorylating multiple substrate proteins and enzymes. The effect of sphingosine, the backbone moiety of sphingolipids, on PKC activity and phosphorylation of endogenous proteins catalyzed by PKC was investigated in nuclei of cow mammary gland. Sphingosine inhibited nuclear PKC activity when lysine-rich histone was used as the substrate. The sphingosine inhibition of the PKC activity was reversed by the excess addition of PS, but not by OAG or Ca2+. Several nuclear proteins, including 56-kDa, 43-kDa, 38-kDa and 36-kDa proteins, were shown to be substrates for PKC. Of the substrate proteins, the 38-kDa and 36-kDa proteins were identified as annexin I, the Ca2+/phospholipid-binding protein; the 56-kDa and 43-kDa proteins have not yet been identified. Sphingosine inhibited phosphorylation of the 56-kDa protein and the 36-kDa annexin I, whereas it enhanced that of the 43-kDa protein. The 38-kDa annexin I species was unaffected by sphingosine. As with the PKC activity, inhibition by sphingosine of phosphorylation of the 56-kDa protein and 36-kDa annexin I was reversed by the excess addition of PS, but not by OAG or Ca2+. In addition, by the excess addition of PS and not by OAG or Ca2+, the sphingosine-enhanced phosphorylation of the 43-kDa protein was reversed and returned to near the level in the absence of sphingosine. It is suggested that sphingosine is involved in the regulation of PKC-dependent phosphorylation in the nucleus by modulating the association of PKC or its substrates, particularly annexin I, with membrane phospholipids in cow mammary gland.     

Electrolyte composition of mink (Mustela vison) erythrocytes and active cation transporters of the cell membrane

Red blood cells from mink (Mustela vison) were characterized with respect to their electrolyte content and their cell membranes with respect to enzymatic activity for cation transport. The intra- and extracellular concentrations of Na+, K+, Cl-, Ca2+ and Mg2+ were determined in erythrocytes and plasma, respectively. Plasma and red cell water content was determined, and molal electrolyte concentrations were calculated. Red cells from male adult mink appeared to be of the low-K+, high-Na+ type as seen in other carnivorous species. The intracellular K+ concentration is slightly higher than the extracellular one and the plasma-to-cell chemical gradient for Na+ is weak, though even the molal concentrations may differ significantly. Consistent with the high intracellular Na+ and low K+ concentrations, a very low or no ouabain-sensitive Na+, K(+)-ATPase activity and no K(+)-activated pNPPase activity were found in the plasma membrane fraction from red cells. The Cl- and Mg2+ concentrations expressed per liter cell water were significantly higher in red cells than in plasma whereas the opposite was the case with Ca2+. The distribution of Cl- thus does not seem compatible with an inside-negative membrane potential in mink erythrocytes. In spite of a steep calcium gradient across the red cell membrane, neither a calmodulin-activated Ca(2+)-ATPase activity nor an ATP-activated Ca(2+)-pNPPase activity were detectable in the plasma membrane fraction. The origin of a supposed primary Ca2+ gradient for sustaining of osmotic balance thus seems uncertain.     

植物角质降解菌的种属鉴定、发酵条件优化及酶学性质研究

本试验旨在通过角质降解菌株X8P的种属鉴定、发酵条件优化和酶学性质研究,探讨降解植物表面角质层,进一步改善动物对植物纤维利用的可能新方案。试验通过形态观察和16 S r DNA测序鉴定菌株X8 P种属,并对其产酶所需碳源、氮源、发酵温度与时间进行优化,其发酵液经硫酸铵盐析沉淀获得其胞外蛋白粗酶,并对其粗酶催化的适宜p H和p H稳定性、温度和温度稳定性,以及有机溶剂、表面活性剂和金属离子对其活性的影响进行研究。结果表明:1)菌株X8P经形态观察和分子鉴定为东方醋杆菌(Acetobacter orientalis)。2)菌株X8P适宜产酶发酵条件为溶菌肉汤(LB)培养基中37℃发酵4 d,1%橄榄油和1%葡萄糖明显促进菌株产酶,而1%可溶性淀粉明显抑制菌株产酶。3)该菌株胞外粗酶催化适宜p H和温度分别为6.5和45℃,且表现出一定p H稳定性,但在有机溶剂中不稳定,仅甘油中可保留全部活性,在二甲基亚砜(50%)中活性可保留66%。吐温(Tween)-20(1 mmol/L)、Tween-80(1 mmol/L)和聚乙二醇辛基苯基醚(1和10 mmol/L)可使菌株X8P粗酶活性提高3%~35%。金属离子钾离子(K+)、锰离子(Mn2+)(1和10 mmol/L)可使菌株X8P粗酶活性提高2%~20%。由此可见,菌株X8P具有一定的产角质酶潜力和应用前景,可进一步深入研究。     

柞蚕幼虫在体脑神经节自发放电活动的初步研究

以柞蚕 5龄幼虫为材料 ,运用微电极电生理技术 ,以细胞外记录方法记录了柞蚕幼虫在体脑神经节的自发放电活动 ,并观察了胞外Na+、K+、Ca2 +的浓度变化对放电活动的影响。结果表明 ,柞蚕幼虫脑神经节的胞外放电活动有 3种形式 ,其离子机制可能既有Na+动作电位 ,又有Ca2 +动作电位。     

Activity of supplemental enzymes and their effect on nutrient utilization and growth performance of growing chickens as affected by pelleting temperature.

Activity of supplemental enzymes in a barley-soybean-maize based diet at 60, 75 and 90 degrees C pelleting temperatures was studied using feed viscosity, in-vitro enzyme activity and broiler performance data. High pelleting temperatures increased feed viscosity but supplemented enzymes reduced the viscosity at all three temperatures levels by 11, 14 and 17%, respectively. Water intake and losses in excreta of birds were found to be affected by feed viscosity. Activity of cellulase enzyme, measured using the radial diffusion method, was unaffected at 60 and 75 degrees C, but reduced by 73% in feed processed at 90 degrees C. Enzymes increased the weight gain of broilers by 11.1% at 90 degrees C, but no effect could be seen at low pelleting temperatures possibly due to high dietary protein and energy contents. Feed intake was unaffected by enzymes. Birds consumed 6% more feed and grew 9% faster when the pelleting temperature was increased from 60 to 75 degrees C. Reduced feed intake and daily weight gain observed at 90 degrees C could be fully compensated by the enzyme supplementation. High pelleting temperature reduced energy metabolizability (3.2%) and nitrogen utilization (4%) but enzyme almost compensated them (by 3.3% and 2.6%, respectively). No interaction could be detected between the pelleting temperatures and enzymes. It is concluded that pelleting temperatures as high as 90 degrees C drastically reduce cellulase activity, energy and nitrogen utilization thus lowering broiler performance. Either the remaining activity of cellulase or other thermostable enzymes can prevent the losses.