Purification and properties of exo-inulinases from Penicillium janczewskii growing on distinct carbon sources

Penicillium janczewskii, isolated from the rhizosphere of Vernonia herbacea, grows rapidly on media containing either sucrose or inulin, although inulin more than sucrose induced the production of inulinases. Three different extracellular beta-fructofuranosidases (two inulinases and one invertase) were purified from fungal cultures grown on sucrose or inulin, through precipitation with ammonium sulfate, and anion-exchange, hydrophobic interaction and gel filtration chromatographies. The optimum temperature of the three enzymes was approximately 60 C, optimum pH 4-5.5 and apparent molecular mass of 80 kDa. K(m) and V(max) values determined for invertase on sucrose were respectively 3.7 10(-4) M and 7.9 10(-2) micromol/min/mL, and on inulin 6.3 10(-2) M and 2.09 10(-2) micromol/min/mL. The values of k(m) for the two inulinases were 8.11 10(-4) and 2.62 10(-3) M, being lower for inulin when compared to those obtained for sucrose. The inulinases did not produce oligofructans from inulin, indicating they are primarily exoinulinases. The differences found in inulinase induction patterns when inulin or sucrose was used seem to be related to modifications on the enzyme properties, mainly concerning substrate affinity.     

The β-fructofuranosidase activities of a strain of Clostridium acetobutylicum grown on inulin

Summary The -fructofuranosidase activities of a strain of Clostridium acetobutylicum, selected for its capacity to grow on inulinic substrates, were investigated. When grown on inulin, this strain produced extracellular and intracellular -fructofuranosidases, both of which hydrolysed inulin (inulinase activity) and sucrose (invertase activity). Inulinase activity was higher than invertase activity in the extracellular preparation, the opposite being observed for the cellular preparation. The effects of pH and temperature, substrate specificity and the kinetic constants for inulin and sucrose were studied on both preparations, as well as induction by inulin and repression by glucose and fructose of inulinase and invertase activities. The overall results were consistent with the existence of a least one inulinase, (EC 3.2.1.7), mainly but not entirely released in the extracellular medium, and an invertase (3.2.1.26) localized within the cell.Time course hydrolysis experiments of dalhia inulin and Jerusalem artichoke inulofructans by extracellular inulinase showed that this preparation had a remarkably high specificity for hydrolysis of long chain inulofructans.     

Purification and some properties of extracellular invertase B from Zymomonas mobilis

Zymomonas mobilis is a Gram-negative ethanologen that can ferment glucose, fructose, and sucrose. Three enzymes that hydrolyze sucrose were found in a zymogram of electrophoretically separated proteins of Z. mobilis CP4. Two were invertase,, Inv A and Inv B; the latter was studied. Inv B is extracellular and accounts for at least 60% of the saccharolytic activity found in the culture broth of Z. mobilis CP4. The enzyme was purified 51-fold in 17% yield from culture broth of Z. mobilis grown on sucrose. It is a -fructosidase, monomeric with a molecular mass of 47 kDa and pI of 4.3. Its K _m for sucrose is 86 mm, and it has high catalytic activity (V _max = 1800 mol product/min per milligram protein). The purification and some properties of Inv B are presented. Correspondence to: D. E. Eveleigh     

Microbial enzymatic production and applications of short-chain fructooligosaccharides and inulooligosaccharides: recent advances and current perspectives

The industrial production of short-chain fructooligosaccharides (FOS) and inulooligosaccharides is expanding rapidly due to the pharmaceutical importance of these compounds. These compounds, concisely termed prebiotics, have biofunctional properties and hence health benefits if consumed in recommended dosages. Prebiotics can be produced enzymatically from sucrose elongation or via enzymatic hydrolysis of inulin by exoinulinases and endoinulinases acting alone or synergistically. Exoinulinases cleave the non-reducing β-(2, 1) end of inulin-releasing fructose while endoinulinases act on the internal linkages randomly to release inulotrioses (F3), inulotetraoses (F4) and inulopentaoses (F5) as major products. Fructosyltransferases act by cleaving a sucrose molecule and then transferring the liberated fructose molecule to an acceptor molecule such as sucrose or another oligosaccharide to elongate the short-chain fructooligosaccharide. The FOS produced by the action of fructosyltransferases are 1-kestose (GF2), nystose (GF3) and fructofuranosyl nystose (GF4). The production of high yields of oligosaccharides of specific chain length from simple raw materials such as inulin and sucrose is a technical challenge. This paper critically explores recent research trends in the production and application of short-chain oligosaccharides. Inulin and enzyme sources for the production of prebiotics are discussed. The mechanism of FOS chain elongation and also the health benefits associated with prebiotics consumption are discussed in detail.     

Molecular weights,poly(A)-content,and partial separation of chick globin mRNAs

Poly(A)-containing 9S RNA from chick reticulocytes was electrophoresed on formamide-polyacrylamide gels. The molecular weight was determined to be 211 000±10 000 daltons. The RNA was separated into three different fractions with respect to molecular weight. These RNAs were translated in a wheat germ cell-free system. The lower molecular weight RNA directed up to 95% -chain synthesis, compared to 60% for the higher molecular weight RNA. This was accompanied by a relative increase for -chain synthesis with increasing molecular weight. It could also be shown by hybridization with labelled poly(U) that the average poly(A) length decreased from about 83 nucleotides for fraction I to 36 nucleotides for fraction III. Our results suggest that fractionation of avian 9 S globin mRNA by electrophoresis on formamide-polyacrylamide gels is dependent upon two parameters, namely differences in the lengths of the non-poly(A)-containing portion of the and mRNAs and differences in the poly(A) lengths.     

Immobilization studies of whole microbial cells on transition metal activated inorganic supports

Summary Whole cells of Saccharomyces bayanus, Saccharomyces cerevisiae and Zymomonas mobilis were immobilized by chelation/metal-link processes onto porous inorganic carriers. The immobilized yeast cells displayed much higher sucrose hydrolyzing activities (90–517 U/g) than the bacterial, Z. mobilis, cells (0.76–1.65 U/g). The yeast cells chelated on hydrous metal oxide derivative of pumice stone presented higher initial -d-fructofuranosidase (invertase, EC 3.2.1.26) activity (161–517 U/g) than on other derivatives (90–201 U/g). The introduction of an organic bridge between the cells and the metal activator led to a decrease of the initial activity of the immobilized cells, however S. cerevisiae cells immobilized on the carbonyl derivative of titanium (IV) activated pumice stone, by covalent linkage, displayed a very stable behaviour, which in continuous operation at 30° C show only a slightly decrease on invertase activity for a two month period (half-life=470 days). The continuous hydrolysis of a 2% w/v sucrose solution at 30° C in an immobilized S. cerevisiae packed bed reactor was described by a simple kinetic model developed by the authors (Cabral et al., 1984a), which can also be used to predict the enzyme activity of the immobilized cells from conversion degree data.     

The relationship between sucrose hydrolysis,sorbitol formation and mineral ion concentration during bioethanol formation using Zymomonas mobilis 2716

Summary Investigations into the relationship between sucrose hydrolysis, sorbitol formation and mineral ion concentration during bioethanol formation by Zymomonas mobilis 2716 revealed two distinct phenomena responsible for carbon flow diversion, a sucrose effect and a salt effect. Neither of the two phenomena affects sucrose hydrolysis, but they divert carbon flow of the fructose monomer leading to its own accumulation, sorbitol or oligosaccharide formation. Sucrose concentrations in excess of 15% (w/v) led to sorbitol formation, the level of which may exceed 2% (w/v) depending upon glucose accumulation during sucrose hydrolysis. Increasing mineral ion concentrations led initially to carbon losses and finally to fructose accumulation instead of sorbitol formation. This carbon loss can be corrected by the addition of invertase, which in turn leads to an increase in sorbitol, fructose and ethanol. Potassium and chloride are the dominant ions responsible for suppression of sorbitol formation and fructose uptake, encouraging oligosaccharide formation. These fructooligosaccharides must be of a type which can be converted to fructose, sorbitol and ethanol through the action of invertase. The requirement of invertase addition to prevent fructooligosaccharide formation is indirect evidence that Z. mobilis 2716 does not produce invertase.Offprint requests to: H. W. Doelle     

Structural studies of an Inv (1, −2) kappa light chain

A Bence Jones protein with phenotype Inv (1, –2) was isolated from the urine of a patient with multiple myeloma. Inv typing of the patient's relatives established the presence of anInv ¹ allele in the kindred, and that the patient's genotype wasInv ¹/Inv ³. Hence, the absence of Inv (2) in the Bence Jones protein was shown to be genetic and not an artifact caused by the disease. The tryptic peptide-containing residues 191 through 194 were isolated and shown to be composed of Leu, Tyr, Ala, Cys, with Leu at the amino end. Hence, the residue at 191 is the same as that present in Inv (1, 2) Bence Jones proteins. More detailed study of the tryptic peptides established that residue 153 is Val rather than Ala as in all other K chains thus far studied. The primary sequence: Ala¹⁵³, Leu¹⁹¹ determines Inv (1, 2); Ala¹⁵³, Val¹⁹¹ determines Inv (3); and Val¹⁵³, Leu¹⁹¹ determines Inv (1). The Val¹⁵³, Val¹⁹¹ sequence has not been observed. It may correspond to Inv (–). These data are strikingly similar to the data for the Kern and Oz isotypes (changes at 154 and 191, respectively) in the chain. As in the case of theK chain, only three of the four possible combinations have been observed. The implications of this parallelism and of crystallographic findings on chains, reported by others, are discussed.     

Sucrose Concentration in Liquid Media Affects Soluble Carbohydrates,Biomass and Storage Quality of Micropropagated Hosta

The effects of sucrose concentration (1, 3, 5, or 7% w/v) in liquid media, in the presence and absence of benzylaminopurine (BAP), on internal carbohydrate status and growth of Hosta tokudama Tratt. Newberry Gold during the multiplication phase (stage II) was investigated. Cultures from all treatment combinations were transferred to media containing 3% (w/v) sucrose during the rooting phase (stage III). At the end of the stage III, these micropropagules were subjected to 5 weeks of storage at 10 °C under low light (photosynthetic photon flux of 5 µmol m^–2s^–1). Endogenous concentrations of soluble sugars (glucose, fructose, and sucrose) in the plantlets increased linearly as the media sucrose concentration increased from 1% to 7% during stage II. Root and shoot biomass increased with increasing media sucrose concentration. BAP increased the biomass and multiplication rate but did not affect internal concentration of soluble sugars. While in storage, endogenous sugar levels and plantlet dry weight remained unchanged for all treatments. Following storage, plants originally cultured in 5% and 7% media sucrose had higher dry weight and less leaf chlorosis than those cultured in 1% and 3% media. Differences in endogenous soluble sugar levels at the end of stage III rooting, and after storage were related to the sucrose concentration of the initial stage II multiplication medium. Increased media sucrose levels during the multiplication cycle has a positive, long-term effect on plant morphology and quality.     

Sucrose utilization during somatic embryo development in black spruce: involvement of apoplastic invertase in the tissue and of extracellular invertase in the medium

The involvement of apoplastic invertase (Ap Inv) and sucrose synthase (SuSy) in the somatic embryo development of black spruce (Picea mariana) was investigated under different maturation conditions. Replacing 6% sucrose with 3% or 1% sucrose in the maturation medium drastically decreased Ap Inv activity and amount in embryogenic tissues. This was accompanied by a decrease in the hexose pool that resulted in a lower starch deposition and protein amount in embryogenic tissues together with a lower embryo production. Conversely, SuSy activity was stable during maturation regardless of the sucrose concentration used in the medium. The presence of an extracellular enzyme responsible for sucrose hydrolysis in the maturation medium was also verified. An immunodetection experiment with anti-acid invertase antibodies revealed the presence of an active 53 kDa polypeptide in the medium, which had a similar molecular mass to that of the Ap Inv polypeptide found in embryogenic tissues. Utilization of sucrose from the medium by the tissues was also studied using labelled 14C-sucrose. Distribution of the radioactivity between tissular sucrose, glucose, and fructose showed that sucrose was diffused into the cell wall of embryogenic tissues and partly hydrolyzed by Ap Inv. These results show that the utilization of sucrose from the medium, the Ap Inv activity in embryogenic tissues, and the release of an active invertase into the medium operate together for the utilization of the carbohydrates during somatic embryo development in black spruce.     

Molecular Weight Determination of Component Polypeptides of Glutenin after Fractionation by Gel Filtration

Reduced and cyanoethylated glutenin was fractionated into three fractions (F I, F II and F III) by gel filtration on Sephadex G–100 in 0.1 m acetic acid. The molecular weight determination was made with these three fractions by sedimentation equilibrium in 6.5 m guanidine hydrochloride containing 0.01 m acetic acid. The molecular weight obtained was 44,000 for F II, and 32,000 for F III. F I showed a distribution of molecular weight due to the aggregation. The average molecular weight of F I was 52,000, being 27,000 at the meniscus and 98,000 at the bottom. The estimation of molecular weight by SDS–PAGE* gave overestimated values for glutenin polypeptides, as was already reported for gliadin.     

Characterization and localization of three soluble invertase forms from Lilium longiflorum flower buds

Three invertase forms (EC 3.2.1.26) were identified in soluble extracts from developing flower buds of Lilium longiflorum Thunb. cv. Nellie White. The enzymes were separable on a diethylaminoethyl (DEAE)-Sephacel column and designated invertase I. II or III according to the order of elution from Sephacel. To determine tissue specificity of these floral invertases, anthers were separated from tepal. pistil and filament tissue, and analyzed for invertase activity. Invertase I was localized primarily in anthers, with invertases II and III being present in much smaller amounts (less than 5% of the invertase I activity). Much higher levels of invertases II and III were found in the nonanther organs of the flower, where essentially no invertase 1 was detectable. Further purification of each form (using gel filtration. Con-A-Sepharose affinity chromatog-raphy and hydrophobic interaction chromatography on phenyl-agarose) resulted in 135- 189- and 202-fold purification of pooled fractions from DEAE-Sephacel. respectively, and established that each invertase form is a glycoprotein. Each was an acid invertase. with pH optima between 4.0 and 5.0 and an apparent molecular mass of 77 500 Da (as determined by Sephadex gel filtration). The invertases had sucrose K_m values of 1.0. 6.4 and 6.6 m M . and temperature optima of 40. 50 and 45°C. respectively. A temperature stability study revealed that invertase III was the most thermostable, followed by II and I. Invertases II and III had lower affinity to raffinose and stachyose than invertase I. All three enzymes were completely inhibited by Hg²⁺ or Ag⁺ ions at 1.7 m M . At this concentration. Cu^2- showed differential partial inhibition . Although fructan was shown to be present in both anther and nonanther tissues of Lilium flower buds, these invertases showed no sucrose:sucrose fructosyltransferase (EC 2.4.1.99) activity.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

The presence of two serine racemases in Streptomyces garyphalus,a d-cycloserine producer

Two serine racemases (I and II) were isolated from Streptomyces garyphalus. Serine racemase I (molecular weight 93,000) was purified to a single band in an analytical electrofocusing system. Serine racemase II (molecular weight 73,000) was partially purified. Both enzymes used pyridoxal-5-phosphate as cofactor. Besides serine the enzymes utilized alanine as substrate but no other amino acid tested. The K _m values of l-alanine and l-serine for enzyme I were 111 mM and 35 mM respectively. Enzyme I was not inhibited by d-cycloserine but by hydroxylamine. Both substances inhibited enzyme II. The serine racemases may be involved in the biosynthesis of d-cycloserine in S. garyphalus.     

Cloning and characterization of an exoinulinase from Bacillus polymyxa

A gene encoding an exoinulinase (inu) from Bacillus polymyxa MGL21 was cloned and sequenced. It is composed of 1455 nucleotides, encoding a protein (485 amino acids) with a molecular mass of 55522 Da. Inu was expressed in Escherichia coli and the His-tagged exoinulinase was purified. The purified enzyme hydrolyzed sucrose, levan and raffinose, in addition to inulin, with a sucrose/inulin ratio of 2. Inulinase activity was optimal at 35°C and pH 7, was completely inactivated by 1 mM Ag⁺ or Hg²⁺. The K _m and V _max values for inulin hydrolysis were 0.7 mM and 2500 M min^–1 mg^–1 protein. The enzyme acted on inulin via an exo-attack to produce fructose mainly.     

Physiological effect of sugars on various digestive enzymes ofSpodoptera littoralis larvae

Summary Feeding of Styropor lamellae treated with 0.25 M maltose, sucrose, raffinose or melezitose byS. littoralis larvae induced amylase activity to 5–6 fold and invertase and protease activity to 2–3 that of the control. Melibiose induced amylase activity to about 4-fold and invertase and protease activity to 188% and 173%, respectively, that of the control. -lactose stimulated the various digestive enzymes to various extents; cellobiose had practically no effect. Glucose and fructose induced the various digestive enzymes to about the same level but to a much higher extent than galactose.The feeding rate of the various sugar compounds except maltose, correlates well with their stimulating activity on the digestive enzymes. Maltose, which induced the larval digestive enzymes to about the same level as that of sucrose, elicited a considerably lower feeding response. The excretion index of sugars stimulating digestive enzymes and eliciting a feeding response, such as maltose, sucrose, raffinose and melezitose, is considerably lower than that of the other sugars.In a comparative test, sucrose, maltose and raffinose induced the various digestive enzymes to about the same level as their equivalent monosaccharide components. However, -lactose and cellobiose, which have the same components as melibiose and maltose but differ in their configuration, affect the various enzymatic systems weakly.Contribution from The Volcani Center, Agricultural Research Organization, Bet Dagan, Israel. 1972 Series, No. 2171-E. Definitions. Feeding response: express the intake of sugar plus insert Styropor carrier. Excretion index: ratio between weight of fecal pellets and Styropor lamellae consumed.The authors wish to thank Mr. K. R. S. Ascher for fruitful discussions, Dr. A. Genizi for statistical analysis of the results, Mrs. Sara Yablonski and Mrs. Shulamit Cohen for skillful technical assistance, and Mrs. Shoshana Hadad for insect rearing.     

Identification and characterization of inulinases by bioinformatics analysis of bacterial glycoside hydrolases family 32 (GH32)

The glycoside hydrolase family contains enzymes that break the glycosidic bonds of carbohydrates by hydrolysis. Inulinase is one of the most important industrial enzymes in the family of Glycoside Hydrolases 32 (GH32). In this study, to identify and classify bacterial inulinases initially, 16,002 protein sequences belonging to the GH32 family were obtained using various databases. The inulin-effective enzymes (endoinulinase and exoinulinase) were identified. Eight endoinulinases (EC 3.2.1.7) and 4318 exoinulinases (EC 3.2.1.80) were found. Then, the localization of endoinulinase and exoinulinase enzymes in the cell was predicted. Among them, two extracellular endoinulinases and 1232 extracellular exoinulinases were found. The biochemical properties of 363 enzymes of the genus Arthrobacter, Bacillus, and Streptomyces (most abundant) showed that exoinulinases have an acid isoelectric point up to the neutral range due to their amino acid length. That is, the smaller the protein (336 aa), the more acidic the pI (4.39), and the larger the protein (1207 aa), the pI is in the neutral range (8.84). Also, a negative gravitational index indicates the hydrophilicity of exoinulinases. Finally, considering the biochemical properties affecting protein stability and post-translational changes studies, one enzyme for endoinulinase and 40 enzymes with desirable characteristics were selected to identify their enzyme production sources. To screen and isolate enzyme-containing strains, now with the expansion of databases and the development of bioinformatics tools, it is possible to classify, review and analyze a lot of data related to different enzyme-producing strains. Although, in laboratory studies, a maximum of 20 to 30 strains can be examined. Therefore, when more strains are examined, finally, strains with more stable and efficient enzymes were selected and introduced for laboratory activities. The findings of this study can help researchers to select the appropriate gene source from introduced strains for cloning and expression heterologous inulinase, or to extract native inulinase from introduced strains.     

Indole-3-acetic acid content and glutamine synthetase activity in the pericarp, and peroxidase activity and isoenzymes in the meso- and exocarp of growing peach fruits

The indole-3-acetic acid (IAA) content in peach pericarp (Prunus persica L. Batsch cv. Merry) was highest at early stage I of development (200 ng/g fresh wt), decreased to the lowest level during stage II, and rose again at stage III to 60–70 ng/g fresh wt. High activity of glutamine synthetase was found in the pericarp during stage I. The soluble peroxidase activity was highest in the meso- and exocarp at stage II, and isoenzymatic changes in this fraction corresponded to the transition from cationic isoenzymes, predominant at stage I, to anionic isoenzymes at stage III. The ionically bound peroxidase activity in these tissues was highest at stage I. The three developmental stages showed marked differences in auxin content and enzyme activities; for peroxidases these changes reflect a developmental expression pattern for the isoenzymes.     

Genome analysis of intergeneric hybrids of apomictic and sexual Australian Elymus species with wheat,barley and rye: implication for the transfer of apomixis to cereals

Summary Four hybrids were obtained between three Australian Elymus taxa and three cereal grains: wheat, rye, and barley. Mean meiotic metaphase-I configurations were 41.14 I, 0.42 rod II, 0.003 ring II, and 0.01 III for E. scabrus var plurinervis x Triticum aestivum (1 hybrid plant), 22.27 I, 2.63 rod II, 0.06 ring II, and 0.12 III for E. scabrus var scabrus x Secale cereale (4 hybrid plants), and 26.65 I, 0.66 rod II, 0.00 ring II, and 0.01 III for E. scabrus var plurinervis x Hordeum vulgare (13 hybrid plants). The I genome of barley also paired very little in a B_III hybrid of apomictic E. rectisetus x H. vulgare (2 hybrid plants). Megasporogenesis in this B_III hybrid was at least facultatively apomeiotic, with the same sort of nuclear elongation, apomeiotic division, and dyad formation seen previously in E. rectisetus itself. All four hybrid combinations were sterile. While spike morphology in the E. scabrus x T. aestivum and E. scabrus x H. vulgare hybrids were intermediate to their parents, E. scabrus x S. cereale and E. rectisetus x H. vulgare looked like their maternal parents.     

Studies on a beta-fructosidase (inulinase) produced bySaccharomyces fragilis

Summary A study was made of a β-fructosidase, which is produced extracellularly and intracellularly bySaccharomyces fragilis. The enzyme catalyzes the hydrolysis of inulin, bacterial levans, sucrose, and the fructose portion of raffinose, by splitting off terminal fructosyl units. It attacks β-2,1 as well as β-2,6 linkages. The enzyme content of inulin-grown cells is sufficient to allow fermentation of inulin at the same rate as glucose. The ratio of hydrolysis rates with sucrose and inulin was about 25 for the β-fructosidase ofS. fragilis and about 14,000 for invertase.S. fragilis does not contain significant amounts of invertase and it ferments inulin, sucrose and raffinose with the aid of a related, but different enzyme, inulinase. Conditions of growth were established which favor inulinase synthesis. Highest yields were obtained with inulin as the carbon source, and somewhat lower yields with raffinose. Glucose, fructose and sucrose were poor inducers of inulinase. The pH of the medium during growth on inulin had to be in the range where inulinase could act, otherwise growth was tardy and poor. In an inulin containing medium aeration favored enzyme production as a result of stimulation of growth. The inulinase content of the cells in a unit volume was generally greater than that in the culture medium. The intracellular inulinase could be solubilized quantitatively by autolysis. The intra-and extracellular inulinases were concentrated and purified to the same extent. Comparison of the two preparations with respect to substrate specificity, rate of inactivation by heat, pH optima with sucrose (4.2) and with inulin (5.0), and elution patterns from a column of diethylaminoethyl cellulose, indicated that the intra-and extracellular enzymes were identical.