Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures

The cleaning power of detergents seems to have peaked; all detergents contain similar ingredients and are based on similar detergency mechanisms. To improve detergency, modern types of heavy-duty powder detegents and automatic dishwasher detergents usually contain one or more enzymes, such as protease, amylase, cellulase, and lipase. Alkaliphilic Bacillus strains are often good sources of alkaline extracellular enzymes, the properties of which fulfil the essential requirements for enzymes to be used in detergents. We have isolated numbers of alkaliphilic Bacillus that produce such alkaline detergent enzymes, including cellulase (CMCase), protease, α-amylase, and debranching enzymes, and have succeeded in large-scale industrial production of some of these enzymes. Here, we describe the enzymatic properties, genetics, and structures of the detergent enzymes that we have developed. Received: January 22, 1998 / Accepted: February 16, 1998     

Time course of sympathovagal imbalance and left ventricular dysfunction in conscious dogs with heart failure

To elucidate thetime course of sympathovagal balance and its relationship to leftventricular function in heart failure, we serially evaluated leftventricular contractility and relaxation and autonomic tone in 11 conscious dogs with tachycardia-induced heart failure. We determined adynamic map of sympathetic and parasympathetic modulation by powerspectral analysis of heart rate variability. The left ventricular peak+dP/dt substantially fell from 3,364 ± 338 to 1,959 ± 318 mmHg/s (P < 0.05) on the third day and declined gradually to 1,783 ± 312 mmHg/s at 2 wk of rapid ventricular pacing. In contrast, the timeconstant of left ventricular pressure decay and end-diastolic pressureincreased gradually from 25 ± 4 to 47 ± 5 ms(P < 0.05) and from 10 ± 2 to21 ± 3 mmHg (P < 0.05), respectively, at 2 wk of pacing. The high-frequency component(0.15-1.0 Hz), a marker of parasympathetic modulation, decreasedfrom 1,928 ± 1,914 to 62 ± 68 × 10³ms²(P < 0.05) on the third day andfurther to 9 ± 12 × 10³ms²(P < 0.05) at 2 wk. Similar to thetime course of left ventricular diastolic dysfunction, plasmanorepinephrine levels and the ratio of low (0.05- to 0.15-Hz)- tohigh-frequency component increased progressively from 135 ± 50 to 532 ± 186 pg/ml (P < 0.05) and from 0.06 ± 0.06 to 1.12 ± 1.01 (P < 0.05), respectively, at 2 wk ofpacing. These cardiac and autonomic dysfunctions recovered graduallytoward the normal values at 2 wk after cessation of pacing. Thus aparallel decline in left ventricular contractility with parasympatheticinfluence and a parallel progression in left ventricular diastolicdysfunction with sympathoexcitation suggest a close relationshipbetween cardiac dysfunction and autonomic dysregulation duringdevelopment of heart failure.

     

Tissue distribution of immunoreactive mouse extracellular superoxide dismutase

Protein content and mRNA expression ofextracellular superoxide dismutase (EC-SOD) were investigated in 16 mouse tissues. We developed a double-antibody sandwich ELISA using theaffinity-purified IgG against native mouse EC-SOD. EC-SOD could bedetected in all of the tissues examined (lung, kidney, testis, brownfat, liver, adrenal gland, pancreas, colon, white fat, thymus, stomach,spleen, heart, skeletal muscle, ileum, and brain, in decreasing order of content measured as µg/g wet tissue). Lung showed a markedly higher value of EC-SOD than other tissues. Interestingly, white fat hada high content of EC-SOD in terms of micrograms per milligram protein,which corresponded to that of lung. Kidney showed the strongestexpression of EC-SOD mRNA. Relatively strong expression of the mRNA wasobserved in lung, white fat, adrenal gland, brown fat, and testis.Heart and brain showed only weak signals, and no such expression couldbe detected in either digestive organs or skeletal muscle.Immunohistochemically, EC-SOD was localized mainly to connectivetissues and vascular walls in the tissues examined. Deep staining inthe cytosol was observed in the cortical tubular cells of kidney. Theseresults suggest that EC-SOD is distributed systemically inmice and that the physiological importance of this enzyme may be acompensatory adaptation to oxidative stress, particularly in lung andkidney.

     

Enzymatic Properties of a Novel Liquefying α-Amylase from an Alkaliphilic Bacillus Isolate and Entire Nucleotide and Amino Acid Sequences

A novel liquefying α-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein⁻¹, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55°C. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1,548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (α/β)₈ barrel motif in domain A.     

Enzymatic Properties of a Novel Liquefying α-Amylase from an Alkaliphilic Bacillus Isolate and Entire Nucleotide and Amino Acid Sequences

A novel liquefying α-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein⁻¹, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55°C. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1,548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (α/β)₈ barrel motif in domain A.α-Amylase (1,4-α-d-glucan glucanohydrolase [EC 3.2.1.1]) and pullulanase (pullulan 6-glucanohydrolase [EC 3.2.1.41]) are amylolytic enzymes of industrial importance, particularly in the food and detergent industries. We have found and characterized some unique debranching enzymes, such as a high-alkaline pullulanase (2), an alkali-resistant neopullulanase (16), and an alkaline isoamylase (3), from cultures of alkaliphilic Bacillus strains, and these enzymes can be used as effective additives in dishwashing and laundry detergents under alkaline conditions, especially when used in combination with α-amylase. We have also found the first known alkaline amylopullulanase from alkaliphilic Bacillus sp. strain KSM-1378 (4), which is very unique in that it efficiently hydrolyzes the α-1,6 linkages of pullulan, as well as the α-1,4 linkages of various carbohydrates at different active sites (1, 13).Liquefying α-amylases, particularly the Bacillus licheniformis enzyme (BLA) (35), are used widely in technical application fields, such as in bread making, production of glucose and fructose syrup and fuel ethanol from starch materials, and textile treatment. The demand for α-amylase for use in laundry and automatic dishwashing detergents has also been growing for several years (42). However, most of the Bacillus liquefying amylases, such as the enzymes from Bacillus amyloliquefaciens (BAA) and Bacillus stearothermophilus (BSA) (28), including BLA (35), have pH optima of between 5 and 7.5 (44). These neutrophilic enzymes are essentially not good for use in detergents, because the working pH range between 8 and 11 is relevant to washing in detergents (17). Since Horikoshi (15) first reported an alkaline amylase from alkaliphilic Bacillus sp. strain A-40-2, many alkaline amylases have been found in cultures of, for example, Bacillus sp. strain NRRL B-3881 (31), Bacillus sp. strain H-167 (14), Bacillus alcalothermophilus A3-8 (7), and Bacillus sp. strain GM8901 (21). The alkaline amylases from these alkaliphilic Bacillus strains reported to date are all of the saccharifying type, except for the enzymes from Bacillus sp. strain 707 (22, 41) and B. licheniformis TCRDC-B13 (5). However, very limited or no information about enzymatic properties of these two liquefying amylases is available. In this paper, we report the isolation of a novel liquefying α-amylase (LAMY) from cultures of the alkaline amylopullulanase producer Bacillus sp. strain KSM-1378 (13). This enzyme is highly active at alkaline pH compared with those of other liquefying α-amylases reported to date. Furthermore, analysis of the gene for this α-amylase (amyK) indicates that LAMY exhibits low amino acid identity to the reported liquefying α-amylases.     

Down-Regulation of Cyclin F Levels during Nerve Growth Factor-Induced Differentiation of PC12 Cells

Treatment with nerve growth factor (NGF) produces a marked decrease of cyclin F levels in PC12EY cells. This decrease is prevented by the simultaneous addition of K-252a. A smaller decrease is observed when the cells are treated with fibroblast growth factor, but the addition of epidermal growth factor has no comparable effect. Time course studies show that the decrease in cyclin F precedes the changes produced by NGF in the distribution of cells within the cell cycle. The data suggest that cyclin F is involved in NGF-mediated cell cycle events during the differentiation of PC12EY cells.     

INDUCTION OF HEPATOCYTE GROWTH FACTOR IN THE LIVER, KIDNEY AND LUNG FOLLOWING TOTAL BODY IRRADIATION IN RAT

Hepatocyte factor (HGF) has been shown to have a pleiotropic function to act as a potent organotropic factor in the regeneration of injury in various organs, including the liver, kidney and lung. To examine the involvement of HGF in radiation injury, the authors analysed the changes in HGF mRNA and HGF protein levels in the rat organs (liver, lung, kidney) and plasma following 6 Gy of total body irradiation. Expression of HGF mRNA in the liver and kidney increased 6–48 h after total body irradiation and returned to previous values 1 week later. HGF protein levels in lung and liver showed 1.3-2 fold elevations 1–2 weeks after irradiation (P< 0.05). HGF levels in plasma stayed at undetectable levels up to 1 month after total body irradiation. The labelling index determined 2 weeks and 1 month after total body irradiation indicated no enhancement of regeneration. Thus, total body irradiation induced transient HGF elevation in these organs without enhancement of regeneration     

Five Candidate Genes for Hamster Cardiomyopathy Did not Map to the Cardiomyopathy Locus by FISH Analysis

The Syrian cardiomyopathic hamster (BIO14.6), that developsboth muscular dystrophy and progressive cardiomyopathy, is widelyused as an animal model of autosomal recessive cardiomyopathymimicking human hypertrophic cardiomyopathy, and five geneshave been proposed as strong candidates for the cause of cardiomyopathy.We recently mapped the cardiomyopathy locus of the hamster tothe centromeric region of chromosome 9qa2.1-b1 by constructionof a genetic linkage map of the Syrian hamster. Thus, we analyzedthe loci of the five candidate genes, tropomyosin, cardiactroponin T, adhalin, calpain 3 and cardiac myosin binding protein-C,by the FISH method, and found that these genes were mapped onthe distal portion of chromosome 12qa5 and 4pa2 and the proximalportion of chromosomes 9qb7, 1qc1.1 and 1qb3, respectively.These results provide strong evidence that the five candidategenes previously proposed are not related to the hamster cardiomyopathy.     

Total synthesis of glycononaosyl ceramide with a sialyl dimeric Lex sequence

The first total synthesis of glycononaosyl ceramide with a sialyl dimeric Le^x sequence 1 is described. Regio- and stereo-selective glycosylations of sialyl donors 6,7,8 with the suitably protected Le^x trisaccharide acceptors 9,10 were performed to give the expected tetrasaccharides 15 and 21, which were converted into the corresponding donors 20 and 22. Boron trifluoride etherate-promoted glycosylation of 20 with pentasaccharide acceptor 11 afforded regioselectively the expected nonasaccharide 23. After replacing benzyl groups of 23 by acetyl groups, the anomeric acetate was transformed into the -trichloroacetimidate 27. The crucial coupling between 27 and (2S, 3R, 4E-3-O-benzoyl-2-N-tetracosanoylsphingenine 3 was executed to afford completely protected -glycoside 28. Finally, selective cleavage of the methyl ester and N,O-deprotection of 28 gave the target ganglioside 1.Dedicated to Professor Sen-itiroh Hakomori on the occasion of his 65th birthday.