Due to the importance of Brevibacterium linens in the industrial production of enzymes, amino acids and vitamins, the vast majority of research carried out has focused on expression of extra cellular proteins and amino acid biosynthesis. Most research to date on B. linens has demonstrated that the physiological and metabolic activities of the bacterium are significantly strain dependent. The heterogeneity among different stains of B. linens has also been confirmed by studies of DNA-DNA homology and partial 16S rDNA sequence analysis. A large number of questions remain to be answered in many areas of the physiology, metabolism, genetics, and taxonomy of Brevibacterium linens DSM 20158. As far as we are aware, there has not yet been a report on the global analysis of cellular proteins associated with B. linens DSM 20158. It may be because of the unsequenced genome of this strain. However in the present study due to the establishment of the Brevibacterium linens BL2 shotgun genome sequence, we have focused on three aspects of this bacterium in order to understand the physiology and biochemistry of the organism as a whole (i) by optimizing production, purification and characterization of industrially important extra cellular enzymes such as alpha-amylase, protease, lipase and an important amino acid L-Lysine which this strain produces naturally, (ii) by isolation, purification and characterization of respiratory chain complexes (iii) by charting the cellular and extra cellular proteome analysis of Brevibacterium linens DSM 20158 which lacks a sequenced genome by mass spectrometry-driven sequence similarity searches. One factor optimization is time consuming, laborious and does not give information about interactions between various fermentation variables. Therefore, we used a statistical approach to optimize factors that influence the production of extra cellular enzymes and amino acids. We used solid state fermentation due to its preference over submerged fermentation. Various cultivation parameters were optimized using a statistical approach to improve the alpha amylase, protease, lipase and L-lysine yield by Brevibacterium linens DSM 20158. The Plackett-Burman design was used to screen the fermentation variables followed by the optimization of significant parameters by response surface CCD in each case. Using the optimal factors, alpha amylase and protease yield was found to be twofold higher than that obtained in the unoptimized reference medium whereas lipase and L-lysine production was also found to be improved by using statistical IAbstract approach. The closeness of optimized values to experimental values proved the validity of the statistical model. B.linens was found to have a branched electron transport chain (Respiratory chain), in which electrons can enter the respiratory chain either at NADH (complex I) or at complex II level. In the present study, we were able to isolate and purify the complex-II (succinate dehydrogenase), complex III (menaquinole cytochrome c reductase cytochrome c subunit, complex IV (cytochrome c oxidase) and complex V (ATP synthase) of the plasma membrane of Brevibacterium linens strain DSM 20158. Oxidized, reduced and pyridine ferrohemochrome spectra of membrane-bound complex-II, III and IV of this bacterium showed the presence of cytochrome b, cytochrome c and cytochrome aa3 respectively which were further confirmed by the heme staining. The complex II isolated from Brevibacterium linens strain DSM 20158 seems to contain three subunits of 64.8-, 30- and 12- kDa. The enzymatic activity of succinate dehydrogenase showed that it is highly active in this microorganism. The complex III, also known as Menaquinol- cytochrome c reductase cytochrome c subunit, was identified with a single band of about 26 kDa. The complex IV (cytochrome c oxidase) was seen to be composed of two subunits at 62.8-, 32- kDa and was further confirmed by its enzymatic activity. The Complex V (F 1 F o -ATP synthase), essential for ATP generation by oxidative phosphorylation, is isolated, purified and appears to be biphasic in nature during its kinetic studies. Brevibacterium linens DSM 20158 is an industrially important actinobacterium, but the lack of a genome sequence limits the applicability of conventional protein identification methods to the proteome of this bacterium. Although a shotgun genome sequence for the BL2 strain of this microbe, it does not cover the entire scope of its proteome. This study has established the first comprehensive proteomic reference map of B. linens DSM 20158. The present study is carried out first by identification of proteins by homology database MASCOT followed by the advanced approach of de novo genome sequence assembly and MS BLAST to drive the expanding B. linens scope of proteomics. This study will help to enhance the usability of this strain of B. linens in different areas of research in future rather in food industries only.
مولانا افتخار احمد فریدی دینی حلقوں میں یہ خبر افسوس اور رنج کے ساتھ سنی جائے گی کہ ۲۴؍ اکتوبر ۱۹۹۸ء کو ایک پُرجوش اور سرگرم داعی و مبلغ مولانا افتخار احمد فریدی انتقال فرماگئے، اناﷲ وانا الیہ راجعون۔ مرحوم کا وطن مراد آباد تھا، ایک زمانے میں ان کے ہاتھوں میں وہ پرچم بغاوت تھا جس کو سرفروشان وطن نے برطانوی جبر و استبداد کے خلاف بلند کیا تھا مگر پھر ع پھاڑ کر جیب و آستین کر علم جنوں بلند۔ انہیں حضرت مولانا محمد الیاسؒ کی دینی دعوت سے عشق ہوگیا جس کے لیے وہ ملکوں ملکوں کی خاک چھانتے رہے ، کسی حادثہ میں ان کا ایک پیر کاٹ دیا گیا مگر وہ بیساکھیوں کے سہارے ملک کے و طول و عرض کا دورہ کرتے رہے، سفر حج میں مولانا شاہ معین الدین احمد ندوی کا ساتھ ہوگیا تو ان کی بڑی خدمت کی اور ہر طرح کا آرام پہنچایا۔ ایک دفعہ وہ اپنے ایک تبلیغی دورے میں اعظم گڑھ آئے تو شاہ صاحب نے ان کی بڑی پذیرائی کی۔ جناب سید صباح الدین صاحب سے مخلصانہ تعلق رکھتے تھے اور راقم سے بھی خط و کتابت رہتی تھی۔ کئی برسوں سے تبلیغی دوڑ دھوپ کا سلسلہ موقوف ہوگیا تھا، لیکن اسی جذبے سے اصلاحی و دعوتی رسائل اور مواعظ و ملفوظات کے مجموعے شائع کرتے تھے، وہ غیر مسلموں میں بھی اسلام کی تبلیغ و اشاعت کرتے رہتے تھے۔ اﷲ تعالیٰ ان کی بشری لغزشوں سے درگزر فرمائے اور انہیں جنت نعیم میں جگہ دے۔ آمین! (ضیاء الدین اصلاحی، نومبر ۱۹۹۸ء)
Qur’ān is the Words of Allah (SWT). Its interpretation is very difficult job because of the concept that how one can understand the will of Creator. Prophet Muhammad (SAW) was the first exegete of Qur’ān. His companions were the next one. This chain is continuously running till now. In subcontinent, translations of Qur’ān and its exegesis work started in third century Hijrah. Hundreds and Thousands of Qur’ānic exegeses exist in subcontinent in Arabic, Persian, English and Urdu languages. In subcontinent Sir Syed, Modūdī, Farahī, Shabir Uthmānī, are the big names of the field. Everyone has chosen a secluded methodology/principle to interpret the Qur’ān. These principles are known as Usūl-e-Tafsīr. There are many differences among these Usūl, due to personnel mindset and social scenario of different era. The questions that why much diversity exists in these and what are its causes, are being addressed here in this article. On the basis of analytical study, it is found that reason behind this diversity is the concept that exegesis of Quran is based on verbal traditions instead intellectual. Secondly, no one compiled these principles/methods for interpretation of Qur’ān in early centuries. In ninetieth century, due to the challenge of science and Orientalism, some scholars compiled Usūl-e-Tafsīr according to their own understanding and some insisted on traditional continuity.
Leather industries contribute substantially to the environmental pollution due to huge quantum of solid and liquid waste generated. It is a chemical processing industry facing major environmental challenges, with most crucial one of waste minimization. Chemicals used in leather processing steps i.e., tanning are mostly health hazardous posing serious threats to ecosystem. Research is under way to develop alternative low cost, low waste, cleaner process technologies by reducing the pollution and chemical load on the environment. Intensive efforts are being directed towards use of enzymes as plausible alternative technology for soaking, dehairing and bating processes. Use of cheaper, less toxic and biodegradable proteases for dehairing may reduce or completely eliminate the use of sodium sulfide producing equally fine quality leather. Therefore, the aim of this current research work was to produce bacterial proteases that can be used for dehairing goat skin and hides as an alternative environment friendly process that may replace conventional method of using sodium sulfide in larger amounts. In the present study, thirteen indigenous bacterial strains were isolated from soil samples of Pakistan and screened for their proteolytic potential by measuring zone sizes of protein hydrolysis on nutrient agar plates with 1% casein as substrate. Bacterial strains RM1and ASM1 produced largest zone sizes of hydrolysis of 29mm and 22mm respectively on 1% casein agar plates after flooding with 10% TCA in qualitative protease assay and were selected for further studies. Based on morphological, biochemical and 16S rRNA gene analysis these two strains ASM1 and RM1 were taxonomically characterized as Bacillus cereus and Bacillus subtilis with the nucleotide sequences submitted in NCBI database under accession number KJ600795 and KT004404 respectively. Both strains were cultured in shake flasks and different nutritional and cultural parameters were optimized by one factor at a time strategy in the batch experiments for maximum protease activity. Five different media with different protease substrates were assayed for higher enzyme production. Bacillus cereus ASM1 grows best in M4 medium with beef extract as protein substrate with 8.7 U/mg specific activity. The alkaline pH value of 9 was suitable for maximum protease activity along with 1% glucose as C-Source, inoculum size 2% (v/v ratio) at mesophilic temperature range (30-37⁰C). Maximum enzyme production with a specific activity of 9.58 U/mg was observed after 48 hours of incubation at optimized conditions for ASM1. For Bacillus subtilis RM1, maximum proteases production with a specific activity of 9.82 U/mg was achieved in M3 medium with malt extract as main carbon and nitrogen source. Other optimized conditions include 1% dextrose as best C-Source in 1% inoculum size at pH 6-7 after 48h incubation. Crude enzyme from both of the strains produced at optimized conditions of enzyme production was used in dehairing application of goat skin. Results for dehairing suggested that RM1 was having better efficacy than ASM1, showing complete removal of hairs after time of 24 h incubation. ASM1 can be used in degradation of trimmings and shavings from untanned hides but collagenolytic nature makes it unsuitable for dehairing. RM1 shows ability to produce thermo-tolerant proteases in bulk quantity by growing at high temperature up to 55oC as it was isolated from the soil bank of a hot water spring of Kashmir, Pakistan. Enzyme from Bacillus subtilis RM1 was purified by two step purification process after ammonium sulfate precipitation and gel filtration column chromatography. Overall yield of the enzyme was increased up to 7.53 folds with a specific activity of 55.125 U/mg (25%) after purification. Purified enzyme retained its catalytic activity over a broad range of temperature (5°C to 65°C) and pH (5-8). EDTA inhibited the efficiency of enzyme confirming it as a metallo-protease while SDS-PAGE analysis confirmed the size of protease as 28.24 kDa. Purified protease exhibited excellent stability and activity in the presence of polar solvents and detergents. Moreover, it retained significant enzymatic activity for higher concentrations of commercially available detergents suggesting that it could be used both in leather and textile industries with ecological benefits. Taking all together, it can be concluded that protease enzyme of Bacillus subtilis RM1 could be an attractive candidate for dehairing. Only about 20-25% of wet salted hides/skins are converted into commercial leather, while large percentage is lost in wasteland as leather shavings, buffing dust, trimmings and splits, fat, soluble proteins and solid suspended pollutants. The harmful effects of the traditional disposal methods for tannery wastes have created a dire need and demand for more cost effective and environmentally acceptable alternatives. The most ideal way to minimize the waste generated is to convert them in to reusable items. This has motivated the research community to search for more innovative uses and applications of tannery wastes. As the wastes are highly proteinaceous, a wide range of value added products can be retrieved making the leather industry as an integrated business with value creation opportunities. Present work also deals with the aim of developing an efficient and value-added process for the management of untanned trimmings, produced in large quantities during the pre-tanning operations. The objective is to extract collagen proteins from tannery waste, treat and to convert the isolated collagen into useful products. For this grounded limed, delimed-bated and alkali treated trimmings were dissolved to make a collagen solution using pepsin. The physico-chemical properties of the resultant sponges and effects of the processing steps on the sponges prepared from un-tanned hides were studied. Results showed that bating and alkaline treatments given to proteinaceous fiber networks after liming have considerable effects on the resultant properties of sponges. SDS-PAGE analysis showed the molecular weights of all three were maintained in the range as of a commercial Type 1 collagen i.e., 100-200kDa. Bating appears to be the best treatment among different treatments as it yields the sponges with best characteristics of water absorption and visco-elasticity. Also, it has positive effect on reducing the apparent density of sponges, within desirable density ranges. Thus, the preliminary results of this study suggest feasibility of using these biobased collagen sponges prepared from un-tanned hides as absorbants that can be commercially viable.