Annual evolution of fatty acid profile from muscle lipids of the common carp (Cyprinus carpio) in Madagascar inland waters

Annual evolution of muscle lipids fatty acid (FA) from common carp (Cyprinus carpio) has been determined in 2001 through monthly samplings in the reserve pond of Sisaony (SIS series) and Itasy Lake (ITA series) of the Madagascar highlands. Total lipids from muscle were extracted and quantified according to the Bligh and Dyer method. FA identification was performed by GC-MS of FA methyl esters and FA pyrrolidides and led to the identification of 41 FA; routine analyses of FA were made by capillary GC. Principal component analysis (PCA) was performed on the data set to compare FA profiles. Lipid content is low, ranging from 0.91 to 1.73% of wet muscle, with a low stage during the hot season (January-April) and a higher stage during the cold season (July-October). Three FA dominated the FA composition: oleic acid (17.0-21.5%), palmitic acid (13.1-16.1%), and linoleic acid (9.6-13.2%). Polyunsaturated fatty acids (PUFA) were present in appreciable amounts: arachidonic acid (AA; 2.9-5.9%), docosahexaenoic acid (DHA; 2.9-6.7%), eicosapentaenoic acid (EPA; 1.9-3.4%), and docosapentaenoic acid (DPA; 1.9-4.3%). Two opposite evolution schemes appear within two groups of FA; on the one hand PUFA (both n-3 and n-6 series) show a maximum in August-October and a minimum in January-April, and, on the other hand, oleic, palmitic, and linoleic acids show the opposite maxima and minima. PCA results give confirmation of these evolution schemes, the two groups of FA giving opposite high factor loadings on axis 1. The SIS and ITA series are differentiated by axis 2 by mean of minor FA, mostly odd- and branched-chain. Results indicate that common carp, the second most abundant freshwater fish in Madagascar highlands waters, may be an interesting source of dietary PUFA.     

Purification and characterization of cathepsin L from the muscle of silver carp (Hypophthalmichthys molitrix)

Cathepsin L in silver carp musle was purified to 48.4-fold by acid-heat treatment and ammonium sulfate fractionation, followed by a series of chromatographic separations. The molecular mass of the purified enzyme was 30 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was activated by dithiothreitol and cysteine while it was substantially inhibited by E-64 and insensitive to PMSF and pepstatin A, suggesting that the purified enzyme belongs to a family of cysteine proteinase. Consistent with this conclusion, Zn2+, Cu2+, Co2+, Ni2+, and Fe2+ could strongly inhibit the activity of this enzyme. The optimal pH and temperature were 5.0 and 55 degrees C, respectively. The enzyme catalyzed the hydrolysis of Z-Phe-Arg-MCA with a parameter of K(m) (8.27 microM) and K(cat) (28.7 s(-1)) but hardly hydrolyzed Z-Arg-Arg-MCA, Arg-MCA, and Boc-Val-Leu-Lys-MCA. The microstructure analysis by scanning electron microscopy showed that this proteinase is capable of destroying the network structure of silver carp surimi gels. The enzyme exhibited a higher hydrolytic activity on surimi protein at 65 degrees C than at 40 degrees C.     

Expression of soluble form carp (Cyprinus carpio) ovarian cystatin in Escherichia coli and its purification

A DNA encoding thioredoxin-mature carp ovarian cystatin (trx-cystatin) fusion protein was ligated into a pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3) expression host. After induction by isopropyl beta-D-thiogalactopyranoside, a high level of the soluble form of recombinant trx-cystatin was expressed in the cytoplasm of E. coli. The recombinant trx-cystatin could be purified by Ni(2+)-NTA agarose affinity chromatography. The molecular mass (M) of the recombinant trx-cystatin was approximately 28 kDa composed of recombinant thioredoxin (16 kDa) and recombinant mature carp ovarian cystatin (12 kDa). Both recombinant trx-fused and mature carp ovarian cystatins were stable at pH 6-11. No obvious decrease in activity was observed even after 5 min of incubation at 60 degrees C. They exhibited papain-like protease inhibition activity comparable to that of the mature carp ovarian cystatin, which could inhibit papain and mackerel cathepsins L and L-like, but not cathepsin B.     

Purification, characterization, and cDNA cloning of a myofibril-bound serine proteinase from the skeletal muscle of crucian carp (Carassius auratus)

A myofibril-bound serine proteinase (MBSP) was highly purified from the skeletal muscle of crucian carp (Carasius auratus) by acidic treatment of myofibril solution and chromatographies on Q-Sepharose and benzamidine-Sepharose 6B. MBSP revealed a main protein band of approximately 28 kDa on SDS-polyacrylamide gel electrophoresis (PAGE) and was particularly inhibited by serine proteinase inhibitors. Substrate-specificity analysis revealed that the enzyme specifically cleaved at the carboxyl side of arginine and lysine residues, suggesting the characteristics of a trypsin-type serine proteinase. MBSP gene was cloned on the basis of the N-terminal sequence and the conserved active site peptide of serine proteinases together with 5'-rapid amplification of cDNA ends (5'-RACE) and 3'-RACE. The coding region gave an amino acid sequence of 242 residues including the initiation methionine and a signal peptide of 20 residues. Amino acid residues of His60, Asp106, and Ser196 consisting of the catalytic triad of serine proteinases were conserved in the sequence. Crucian carp MBSP shared relatively high identities with other serine proteinases, especially in well-conserved regions.     

Purification and characterization of calpastatin from grass prawn muscle (Penaeus monodon)

Calpastatin, a specific calpain inhibitor was purified to electrophoretical homogeneity from grass prawn (Penaeus monodon) muscle by 100 degrees C heat-treatment, DEAE-Sephacel, and Q-Sepharose chromatographs. No significant change in the inhibitory activity of crude calpastatin was observed even after 20 min incubation at 100 degrees C, pH 7.0. The purified prawn calpastatin had a molecular weight (M(r)) of 80 and 88.7 kDa determined by SDS-PAGE and Sephacryl S-200 HR gel filtration, respectively. According to the active site titration, the purified calpastatin revealed four beef mu-calpain and two beef m-calpain binding domains, respectively. It was stable during 1 h of incubation at 30 degrees C under pH 4.5-10.0 and shown to be a highly specific inhibitor for calpain.     

Changes in the odorants of boiled carp fillet (Cyprinus carpio L.) as affected by increasing methionine levels in feed

Sixty common carp in groups of five in four tanks per treatment were given three diets containing different increasing amounts of methionine. The aroma extract dilution analysis (AEDA) of the boiled carp fillets resulted in 32 odorants, of which 26 were identified. Ten compounds were quantified using an internal standard (IS), and the very low concentrations of 2-acetyl-1-pyrroline, (Z)-1, 5-octadien-3-one, and methional were calculated by the nasally determined detection limit. The concentration of methional seemed to increase with increasing methionine in the diet. Because the unstable methional could be converted by beta-elimination into methanethiol, the impact resulting in an off-flavor was investigated by headspace analysis. The investigation revealed no difference in the methanethiol contents between the treatments with the lowest and highest methionine supplies.     

In vitro effect of malathion and endosulfan on the LH-induced oocyte maturation in the common CARP,Cyprinus carpio (L.)

Effect of malathion (organophosphorus insecticide) and endosulfan (organochlorine insecticide) on in vitro LH-induced oocyte maturation was investigated in the oocytes of common carp, Cyprinus carpio. In control incubation, LH at the concentration of 10 μg mL^?1 induced 41.2 ± 1.6% of germinal vesicle breakdown (GVBD). When the oocytes were incubated with malathion at the concentrations of 1000, 500, 100, and 50 ppb together with LH it could induce only 13.4 ± 0.4, 14.0 ± 1.0, 12.9 ± 3.5, and 18.1 ± 3.9% of GVBD, respectively. Similarly, when the oocytes were incubated with endosulfan at the concentrations of 0.5, 0.1, 0.05, and 0.01 ppb together with LH, it induced only 12.8 ± 1.6, 8.8 ± 1.2, 20.9 ± 2.1 and 26.0 ± 2.2% of GVBD, respectively. The significance of the result obtained were discussed on the basis of available literature.     

利用不同基因转移方法构建转基因红鲤

本实验研究了大黄鱼肌肉生长抑素前肽基因对红鲤的促生长作用。分别通过RT-PCR和PCR从大黄鱼(Larimichtys crocea)和pIRES-EGFP质粒扩增得到了肌肉生长抑素(MSTN)前肽基因及核糖体内部进入位点序列(IRES)-增强型绿色荧光蛋白基因(EGFP)片段,经测序验证正确后,构建了Tol2转座子供体质粒pT2AL200R150G-MSTN propeptide-IRES-EGFP。通过精子介导法(S1、S2组)、电穿孔法(E1、E2组)及基因枪法构建转基因红鲤(Cyprinus carpio),孵化72h后的鱼苗经荧光显微镜检测,EGFP表达阳性率为:精子介导法S1组38%,精子介导法S2组48%,电穿孔E1组47%,电穿孔E2组53%,基因枪组2%;孵化10d的仔鱼RT-PCR检测EGFP和MSTN前肽基因阳性率为:精子介导法S1组35%,精子介导法S2组45%,电穿孔E1组45%,电穿孔E2组55%,基因枪组1.8%。孵化后75d转基因红鲤与对照组相比,体长和体重分别提高了21.31%和27.59%。本实验结果表明,精子经高渗、低渗保存剂处理并通过电穿孔作用可大幅提高基因转移效率。     

鲤鱼三、四核苷酸重复微卫星座位的筛选及特征分析*

采用磁珠富集法结合放射性同位素杂交法分离出鲤鱼(Cyprinus carpio)三、四核苷酸重复的微卫星阳性克隆1248个。对其中的384个克隆进行测序分析,共得到微卫星序列325个,其中完美型244个（75.08%）,非完美型59个（18.15%）,混合型22个（6.77%）。依据得到的微卫星序列,用Primer 3软件在线设计并合成引物145对,检测结果显示71.43%（80对/112对）的引物在野生个体间表现出多态性,等位基因数在3~12之间,多态信息含量在0.2109~0.8607之间,80%的位点处于高度多态水平。对群体的遗传结构评估结果显示,四核苷酸重复的微卫星标记在黑龙江鲤野生群体表现出的多态性水平（PIC=0.7740）高于三核苷酸重复（PIC=0.5161）和双核苷酸重复（PIC=0.49）,适用于群体间遗传差异分析和遗传连锁图谱的构建。     

盐碱池塘养殖鲤肠道菌群的分子分析

为了了解盐碱池塘养殖鲤肠道细菌群落组成及多样性,提取鲤(Cyprinus carpio L.)肠道细菌基因组DNA,选用细菌通用引物对16S rRNA基因进行了PCR扩增,构建了细菌16S rRNA基因克隆文库。通过对阳性克隆子进行限制性片段长度多态性分析(RFLP),选出有代表性的克隆子进行测序、BLAST比对分析和构建系统发育树。本研究从16S rRNA基因文库中共筛选出176个阳性克隆,经RFLP分析得到28个不同分类操作单元(operational taxonomic unite,OTU)(GenBank登录号:JX262557~JX262584),文库覆盖度为88.6%。16S rRNA序列系统发育分析发现,盐碱塘养殖鲤肠道细菌归属于变形细菌门(Proteobacteria)(包含Alpha和Gamma亚群)、厚壁细菌门(Firmicutes)、拟杆菌门(Bacteroidetes)和梭杆菌门(Fusobacteria)4个门,分别占克隆总数的89.9%、7.9%、1.1%和1.1%。其中,变形细菌门的α-变形菌纲(占克隆总数的88.1%)为优势类群,气单胞菌属为优势菌属。本研究揭示了盐碱池塘养殖条件下健康鲤肠道细菌群落组成。     

Purification and characterization of amylases from small abalone (Sulculus diversicolor aquatilis)

Amylases II-1 and II-2 with molecular weights of 55.7 and 65 kDa, respectively, were purified to electrophoretical homogeneity from small abalone (Sulculus diversicolor aquatilis) by ammonium sulfate fractionation, Sepharose CL-6B, CM-Sepharose CL-6B, and Sephacryl S-100 chromatographs. They had optimal temperatures of 45 and 50 degrees C and an optimal pH of 6.0. The purified amylases were stable at pH 5.0-8.0 and 6.0-8.0, respectively. They were completely or partially inhibited by Hg(2+), Cu(2+), Cd(2+), Zn(2+), iodoacetamide, phenylmethanesulfonyl fluoride, and N-ethylmaleimide, suggesting the existence of cysteine at their active sites. Digestion tests against various polysaccharides suggested that the purified amylases II-1 and II-2 are neoamylases which can hydrolyze both alpha-1,4 and alpha-1,6 glucosidic bonds. Amylase II-2 might be an exo- and II-1 an endo-/exo-amylase.     

Purification and characterization of a carboxypeptidase from squid hepatopancreas (Illex illecebrosus)

The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.     

Purification and characterization of trypsin from the spleen of tongol tuna (Thunnus tonggol)

Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 degrees C. It was stable in a wide pH range of 6-11 but unstable at the temperatures greater than 50 degrees C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0-30%). The enzyme had a Km for Nalpha-p-tosyl-L-arginine methyl ester hydrochloride of 0.25 mM and a Kcat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins.     

Purification and characterization of trimethylamine-N-oxide demethylase from jumbo squid (Dosidicus gigas)

Trimethylamine-N-oxide demethylase (TMAOase) was purified from Jumbo squid (Dosidicus gigas) and characterized in detail herein. The TMAOase was extracted from squid with 20 mM Tris-acetate buffer (pH 7.0) containing 1.0 M NaCl, followed by acid treatment and heat treatment. Then it was purified by deithylaminoethyl-cellulose and Sephacryl S-300 chromatography, subsequently resulting in an 839-fold purification. The molecular mass of the TMAOase was defined to be 17.5 kDa. The optimum pH of the purified TMAOase was 7.0, and its optimum temperature was confirmed to be 55 degrees C. The TMAOase was stable to heat treatment up to 50 degrees C and stable at pH 7.0-9.0. Reducing agents such as DTT, Na2SO3, and NADH were effective at activating TMAOase, and ethylenediaminetetraacetic acid, as well as Mg2+ and Ca2+, could also enhance the activity of TMAOase remarkably, whereas the TMAOase could be significantly inhibited by tea polyphenol, phytic acid and acetic acid. In addition, the TMAOase converted TMAO to dimethylamine and formaldehyde stoichiometrically with a K(m) of 26.2 mM.     

Purification and characterization of the 7S vicilin from Korean pine (Pinus koraiensis)

Pine nuts are economically important as a source of human food. They are also of medical importance because numerous pine nut allergy cases have been recently reported. However, little is known about the proteins in pine nuts. The purpose of this study was to purify and characterize pine nut storage proteins. Reported here is the first detailed purification protocol of the 7S vicilin-type globulin from Korean pine (Pinus koraiensis) by gel filtration, anion exchange, and hydrophobic interaction chromatography. Reducing SDS-PAGE analysis indicated that purified vicilin consists of four major bands, reminiscent of post-translational protease cleavage of storage proteins during protein body packing in other species. The N-terminal ends of vicilin peptides were sequenced by Edman degradation. Circular dichroism (CD) and differential scanning calorimetry (DSC) analyses revealed that pine nut vicilin is stable up to 80 degrees C and its folding-unfolding equilibrium monitored by intrinsic fluorescence can be interpreted in terms of a two-state model.     

草鱼消化道碱性蛋白酶的提取及鉴定

用丙酮将草鱼肠脱脂,磷酸盐缓冲溶液提取粗酶液,再用10%~40%饱和度的硫酸铵分级沉淀制取粗草鱼消化道碱性蛋白酶。粗草鱼碱性蛋白酶的比活力为2 800 U/g,最适作用温度为37°C,最适作用pH为10.3。在50°C保温30 min后还保留60%以上的酶活力,要使其损失约80%的活力,需要在60°C保温30 min。50 mmol/L的甲苯磺酰赖氨酸氯甲酮(TLCK)、甲苯磺酰氟(PMSF)和50μmol/L大豆胰蛋白酶抑制因子(SBTI)分别抑制78.92%、75.98%和80.23%的酶活力。SDS-底物-聚丙烯酰胺凝胶电泳表明碱性蛋白酶的活性成分有4种,一种为非丝氨酸蛋白酶,相对分子质量为105 000;另外三种为胰蛋白酶,相对分子质量分别为26 400、30 750和43 000。     

筛选杂交鲤亲子鉴定的微卫星标记(英文)

微卫星的亲子鉴定技术既能减少分池饲养带来的遗传性状的差异又能减少其所占用饲养池的数量和管理强度,在水产养殖和品种选育中得到了广泛的应用。本研究应用21对微卫星标记对鲤(Cyprinus carpio)杂交家系(49-同胞家系:德国镜鲤(C.carpio var.mirror)♀×荷包红鲤(C.carpio var.wuynanensis)♂;24-同胞家系:荷包红鲤♀×德国镜鲤♂)进行遗传多样性及遗传特性的评价,并检测影响微卫星标记鉴定准确性的几个特性。研究结果标明,(1)筛选出的13对标记:在49-同胞家系中,平均有效等位基因数(K)、期望杂合度(He)、观测杂合度(Ho)和多态信息含量(PIC)分别为0.505、0.648、0.791和0.5889;在24-同胞家系中,K、He、Ho和PIC分别为0.548、0.670、0.819和0.6138。(2)筛选出的13对标记:在49-同胞家系和24-同胞家系中的联合排除率(CPE)分别达到0.999997978和0.999999583。(3)在两个杂交家系中,4个核心位点(MFW29,HLJ392,HLJ044,HLJ855)的联合排除率均高于99%。(4)三种情况下(一个亲本已知,NE-1P;另一个亲本已知,NE-2P;双亲都已知,NE-PP;)的联合非父排除率在49全胞家系中分别为1.34E-02、3.91E-04和2.02E-06;在24全胞家系中分别为1.34E-02、3.91E-04和2.02E-06。(5)模拟分析显示(置信度为99%),当使用11个或更多标记时,在49全胞家系中亲本与子代的配对率为100%;当使用9个或更多标记时,在24全胞家系中亲本与子代的配对率大于98%。(6)UPGMA聚类图表明:在混养条件下,筛选出的13对标记可以明显地将子代与无亲缘关系的家系区分开。本研究筛选出13对微卫星标记完全适用于荷包红鲤与德国镜鲤杂交家系的鉴定,为品种选育提供有效的鉴定工具。     

Purification and characterization of collagenolytic proteases from the hepatopancreas of northern shrimp (Pandalus eous)

Three gelatinolytic proteases (A1, A2, and B) were purified using a synthetic substrate, DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, from the hepatopancreas of Northern shrimp (Pandalus eous) by several chromatographic steps involving hydroxyapatite column chromatography, gel filtration on Superdex75, and ion-exchange chromatography on a MonoQ column. Collagenolytic proteases A2 and B, but not protease A1, were demonstrated to digest native porcine type I collagen at 25 degrees C and pH 7.5. Further characterizations of these two collagenolytic proteases showed that the pH optimum of enzyme A2 against DNP-peptide was found to be 11, whereas that of enzyme B was 8.5. The optimum temperature ranged between 40 and 45 degrees C for both enzymes, although enzyme B appeared to be thermally more stable than enzyme A2 at pH 7.5. Both enzymes were strongly inhibited by PMSF and antipain, which suggests that they belong to collagenolytic serine proteases.     

槲皮素对四氯化碳致建鲤肝细胞损伤的保护作用

本研究旨在探讨槲皮素(quercetin,QC)对化学性肝细胞损伤的保护作用。1,1-二苯基-2-苦基苯肼基自由基(1,6-Bis(diphenylphosphino)hexane,DPPH)在本实验中用来测定槲皮素的自由基清除能力。8mmol/L四氯化碳(CCl4)用作体外诱导剂,构建建鲤(Cyprinus carpio)肝细胞损伤模型。原代培养的肝细胞分别用不同浓度的槲皮素(0.05、0.1和0.2 mg/mL)进行前处理,后处理及前后处理,检测培养上清指标酶((谷草转氨酶(aspartate transaminase,GOT)、谷丙转氨酶(alanine transaminase,GPT)、乳酸脱氢酶(lactate dehydrogenas,LDH))及超氧化物歧化酶(superoxide dismutase,SOD))的酶活性及丙二醛(malondialdehyde,MDA)的释放量,实时荧光定量PCR(Real-time quantitative PCR,qRT-PCR)法测定细胞中细胞色素P450 1A(CYP1A)、3A(CYP3A)及白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)及诱导型一氧化氮合酶(iNOS)m RNA表达量,Western blot测定核转录因子-κB(NF-κB)成员c-Rel和p65的蛋白表达量。结果显示,低浓度的QC即可高效地清除DPPH,表明QC具有较强的自由基清除能力。QC可以显著提高细胞活力及SOD活性,且有效地降低了GOT、GPT、LDH和MDA的含量;同时,QC也显著地抑制了CYP1A和CYP3A的表达,对c-Rel和p65及其下游细胞因子的转录也起到了显著的抑制作用。数据统计分析显示,前处理组效果最佳,前后处理组效果次之,后处理组稍差,各组中0.1和0.2 mg/m L槲皮素的抑制效果最明显。综合以上结果,QC对建鲤化学性肝损伤有一定的保护作用。实际生产中,可将槲皮素开发成一种饲料添加剂,以增加水产动物的抗病能力。     

Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings

Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.